We thank Lucile Wrenshall for the use of the plate reader, Nancy Bigley for useful discussion, and Tom Brown for the gift of ICR mice. This work was funded by grant 1R01AI114804 from the National Institute of Allergy and Infectious Diseases and by WSU Faculty Development funds (to J.A.K.). J.N.G. was supported in part by the National Institute of General Medical Sciences grant R25GM090122.

All experiments are carried out in accordance with protocols approved by the Wright State University Lab Animal Care and Use Committee and Institutional Review Board.

NOTE: Human PBMCs are isolated by the Ficoll density gradient centrifugation method5.
1. Spleen Harvest
<ol>
	<li>House adult female ICR mice in standard lab conditions with the food, water, and bedding requirements specific to the species. 
	NOTE: Animals had a mean age of 2.9 months and a mean weight of 30.4 g at the time of euthanasia.</li>
	<li>On the date of isolation (DOI), euthanize the animals individually without other conspecific animals present. Make all efforts to ensure minimal stress to the mouse. Euthanize using CO2 with secondary cervical dislocation to ensure death.
	<ol>
		<li>Place the animal, still inside the transfer cage, into the CO2 chamber and start the gas flow at 5 L/min. This flow rate displaces oxygen at the recommended 10-30% per min.</li>
		<li>After the animal is unconscious, increase the CO2 flow rate to 15 L/min. Check the animal for cessation of breathing and leave in the chamber for an additional 2 min.</li>
		<li>Apply cervical dislocation with distraction force to ensure death.</li>
	</ol>
	</li>
	<li>Harvest the spleen from the animal using aseptic technique within 10 min of death.
	<ol>
		<li>Sterilize two sets of scissors and forceps for the removal of the spleen in an autoclave oven prior to use. Sterilize the dissection surface by applying 70% ethanol.</li>
		<li>Apply 70% ethanol to the mouse abdomen. Using one set of scissors and forceps, make a cut in the fur and skin from the left side of the abdomen, then pull apart and peel back the skin to reveal the peritoneum. Once exposed, apply 4% chlorhexidine solution before opening the peritoneum.</li>
		<li>Using the second set of scissors and forceps, make a 2- to 3-cm cut along the central abdomen. Open the peritoneum and remove the spleen by cutting away visceral attachments and excess fat. Place the spleen in Dulbecco&#39;s phosphate-buffered saline (DPBS).</li>
	</ol>
	</li>
</ol>
2. Preparation of Raw Splenocytes
NOTE: Perform all steps under a laminar flow biosafety cabinet using aseptic technique.
<ol>
	<li>Soak the harvested spleens in 10 ml of deionized sterile H2O for 5 min in a 10 cm culture dish to lyse the surface erythrocytes. 
	NOTE: This step can be omitted if the spleen was damaged during isolation.</li>
	<li>To release the splenocytes, crush the spleens between two sterilized glass slides (frosted side facing the spleen) over a fresh 10 cm culture dish. Mix with 10 ml of RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 &#181;g/ml streptomycin (below referred to as complete RPMI).</li>
	<li>Filter the cells through a sterile 40 &#181;m nylon cell strainer into a new 10 cm culture dish to remove the connective tissue and debris.</li>
	<li>Lyse the remaining erythrocytes by adding 20 ml of RBC lysis buffer composed of 155 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA at pH 7.4 and ~300 mOsm/L. 
	NOTE: Osmolality is measured by a freezing point or a vapor pressure osmometer.
	<ol>
		<li>Transfer the cells from the 10-cm plate to a 50 ml conical tube and centrifuge at 250 x g for 10 min (4 &#176;C).</li>
		<li>Remove the supernatant, add 20 ml of RBC lysis buffer, and re-suspend the pelleted cells by pipetting.</li>
		<li>Incubate at room temperature in the lysis buffer for 10 min with gentle rocking.</li>
		<li>Centrifuge for 10 min at 250 x g (4 &#176;C) to pellet the cells. Pour off the lysis buffer.</li>
		<li>Re-suspend cells in 10 ml of complete RPMI and centrifuge again (250 x g, 10 min, 4 &#176;C). Discard the supernatant.</li>
	</ol>
	</li>
	<li>Re-suspend the splenocytes in 2 ml of complete RPMI warmed to 37 &#176;C for transfer to the nylon wool fiber column.</li>
</ol>
3. Purification of T Lymphocytes
<ol>
	<li>Wash the nylon wool columns twice with 5 ml of complete RPMI. Incubate at 37 &#176;C in a 5% CO2 cell culture incubator for 1 hr. 
	NOTE: It is imperative to keep the nylon wool wet for the duration of the experiment and to use complete RPMI warmed to 37 &#176;C for all nylon wool isolation steps.</li>
	<li>Add isolated splenocytes to the column and incubate for 1 hr to allow the B cells, fibroblasts, and accessory cells to adhere to the nylon wool.
	<ol>
		<li>Add 2 ml of RPMI containing the splenocytes into the column and pass through until the top of the nylon wool is reached.</li>
		<li>Add 2 ml of complete RPMI warmed to 37 &#176;C on top of the nylon wool and pass the medium through the wool until the liquid level reaches the top surface.</li>
		<li>Add 3 ml of warm complete RPMI to the column to completely cover the nylon wool.</li>
		<li>Incubate the loaded column undisturbed in a 37 &#176;C, 5% CO2 cell culture incubator for 1 hr.</li>
	</ol>
	</li>
	<li>Elute the cells by washing the column twice with 5 ml of complete RPMI. Perform an additional wash of the eluted cells by centrifugation.
	<ol>
		<li>Fill the column to the top 2 times with complete RPMI and allow the solution in the column to flow through into a 50 ml sterile conical tube. 
		NOTE: Avoid tapping or bumping the column, which can dislodge the B cells, accessory cells, and fibroblasts adhering to the nylon wool.</li>
		<li>Collect the flow-through fraction and spin at 250 x g for 10 min (4 &#176;C) to pellet the cells. Wash once with 10 ml of complete RPMI. Pellet the cells again (250 x g, 10 min, 4 &#176;C) and re-suspend in 2 ml of complete RPMI.</li>
		<li>Measure the cell density using a hemocytometer and dilute in complete RPMI to 0.5 x 106 cells/ml.</li>
		<li>Seed 1 or 2 ml aliquots of cells into each well of a 24- or 6-well cell culture plate, respectively, and culture the cells at 37 &#176;C with 5% CO2. 
		NOTE: 1 mM 1,4-dithiothreitol (DTT) can be added to each well at this stage to improve T-cell survival.</li>
		<li>Optional: Confirm the efficiency of the isolation protocol by flow cytometry. This protocol normally yields ~80% T lymphocytes23. 
		NOTE: Nylon wool-purified cells contain approximately 50% CD4+ and 23% CD8+ cells. To purify the CD4+ and CD8+ subpopulations further, a single immunomagnetic depletion step can be performed using eight antibodies and magnetic beads24. Alternatively, purer CD4+ and CD8+ T-cell populations can be isolated by positive or negative selection with specific antibodies.</li>
	</ol>
	</li>
</ol>
4. Activation of T Lymphocytes and Experimental Drug Exposure
<ol>
	<li>Activate T lymphocytes within 24 hr of isolation through the addition of PMA and ionomycin calcium salt or magnetic beads coated with anti-CD3 and anti-CD28 antibodies23,25,26. Add experimental drugs (e.g., CsA, FK506, rapamycin, TRAM-34, and FTY720) at the time of activation. 
	NOTE: Here, PMA concentrations varied from 2 to 250 ng/ml, and the ionomycin concentration was kept at 250 nM. If possible, dilutions should be prepared from stock aliquots of each drug so that the volume added to each well of cells is &#8805;1 &#181;l to ensure reproducibility. The anti-CD3/anti-CD28-coated magnetic beads are added at a 1:1 bead-to-cell ratio. Increasing the number of beads per cell (i.e., the bead-to-cell ratio) will increase the intensity of stimulation25,26. The beads are washed and re-suspended in complete RPMI before their addition to the cells. Note that trypan blue stains the beads.</li>
	<li>Culture the cells for 12-72 hr at 37 &#176;C with 5% CO2 before analyzing with the automated cell counter.</li>
</ol>
5. Automated Cell Counter Data Collection
<ol>
	<li>Before running the sample, make sure that the cells are gently mixed with a serological pipette to avoid clumps. This is particularly important for activated lymphocytes because of their increased adhesiveness.
	<ol>
		<li>For cultures that are activated with anti-CD3/CD28 beads, after pipetting, transfer the sample to a 1.5 ml or 2 ml centrifuge tube. Separate the beads by keeping the tube on a magnet for 1-2 min. Use the supernatant containing the cells for analysis. 
		NOTE: Analyze both resting and activated cells within 1 hr to account for any pre-activation of the resting cells by serum present in the culture medium. After 12 hr of PMA/ionomycin stimulation, a small increase in cell size was detectable (Figure 2). At 72 hr, no significant cell size increase compared to 48 hr was observed (data not shown).</li>
	</ol>
	</li>
	<li>Transfer 1 ml of the cell suspension into a sample cup and run it through the automated cell counter according to the instructions in the manual. 
	NOTE: For each of the trials, a minimum of 1 ml (maximum of 2 ml) of cell culture should be used. The automated cell counter uses a syringe to mix trypan blue with the cell suspension and passes the cell-trypan blue mixture over a field that is imaged and analyzed by the software. The software detects trypan blue-stained cells, draws a circle around each cell, and determines the diameter. 100 images are collected from each sample, and cell viability and sizes are determined. The detection threshold of the cell counter used here has a lower limit of 5 &#181;m.</li>
	<li>Transfer data from each run to a spreadsheet for further analysis.</li>
	<li>Calibrate the device on a regular basis using a 1 ml sample of 6 &#181;m and 8 &#181;m diameter polystyrene beads. 
	NOTE: The actual bead sizes measured by the manufacturer for each batch should be used, not the nominal size. 6 and 8 &#181;m beads are convenient because these diameters are close to the expected diameters of resting and activated T cells (see Figure 1).</li>
</ol>
6. Data and Statistical Analysis
<ol>
	<li>Analyze the data using appropriate statistical and graphing software. 
	NOTE: The spreadsheet for each run contains the number of cells with each diameter (range: 5 &#181;m to 70 &#181;m). The cells above a 17 &#181;m diameter are removed from the analysis to exclude dust particles and small bubbles, which can be read as viable cells by the instrument.</li>
	<li>Perform hypothesis testing using Welch&#39;s t-test for data sets with unequal variance; results are considered statistically significant if p &#60; 0.001. The formula used was, 
	<img alt="Equation 1" src="/files/ftp_upload/55212/55212eq1.jpg" /> 
	 
	where <img alt="Equation 2" src="/files/ftp_upload/55212/55212eq2.jpg" /> and <img alt="Equation 3" src="/files/ftp_upload/55212/55212eq3.jpg" /> are sample means, <img alt="Equation 4" src="/files/ftp_upload/55212/55212eq4.jpg" /> and <img alt="Equation 5" src="/files/ftp_upload/55212/55212eq5.jpg" /> are sample variances, and <img alt="Equation 6" src="/files/ftp_upload/55212/55212eq6.jpg" /> and <img alt="Equation 7" src="/files/ftp_upload/55212/55212eq7.jpg" /> are sample sizes for each data set. The number of degrees of freedom is conservatively estimated using the smaller of the two sample sizes for each comparison. Bar graphs are presented as the mean &#177; SEM.</li>
</ol>
7. Splenic T-lymphocyte Proliferation Assay
<ol>
	<li>Measure T-cell proliferation using an MTS- or MTT-based colorimetric plate reader proliferation assay. 
	NOTE: The assay is based on MTS tetrazolium compound (Owen&#39;s reagent) reduction to soluble formazan product, likely by NADPH or NADH produced in metabolically active cells by dehydrogenase enzymes.</li>
	<li>Count purified T cells with a hemocytometer and re-suspend them in complete RPMI-1640 at a final density of 1 x 106 cell/ml. 
	NOTE: 1 mM DTT can be added to the cells at this stage.</li>
	<li>Activate the cells with PMA and ionomycin along with test drugs, if required, and mix gently (similar to step 4).</li>
	<li>Plate 100 &#181;l of the cells in each well of a 96-well cell culture treated plate and incubate at 37 &#176;C with 5% CO2 for 48 hr. Add 100 &#181;l of RPMI-1640 media without cells into one well to obtain a reading of the background absorbance.</li>
	<li>Add 20 &#181;l of MTS-based reagent to each well and incubate at 37 &#176;C with 5% CO2 for 4 hr.</li>
	<li>Take absorbance measurements at 490 nm using a plate reader. The absorbance measurements correspond to the number of metabolically active cells in each well. 
	NOTE: Subtract background levels if needed. The background absorbance values depend upon the type of culture medium, serum, external pH, and duration of MTS reagent exposure to light. MTS-based reagents are light-sensitive, and exposure to light for several hours may result in higher background absorbance values.</li>
</ol>

<ol>
	<li>Weiss, A., Samelson, L. E., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-lymphocyte+activation.">T-lymphocyte activation.</a> Fundamental Immunology. , 321-364 (2003).</li><li>Gergely, P., Ernberg, I., Klein, G., Steinitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastogenic+response+of+purified+human+T-lymphocyte+populations+to+Epstein-Barr+virus+(EBV).">Blastogenic response of purified human T-lymphocyte populations to Epstein-Barr virus (EBV).</a> Clin Exp Immunol. 30 (3), 347-353 (1977).</li><li>Sanderson, R. J., Rulon, K., Groeneboer, E. G., Talmage, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+murine+splenic+lymphocytes+to+concanavalin+A+and+to+co-stimulator.">The response of murine splenic lymphocytes to concanavalin A and to co-stimulator.</a> J Immunol. 124 (1), 207-214 (1980).</li><li>Decoursey, T. E., Chandy, K. G., Gupta, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+induction+of+ion+channels+in+murine+T+lymphocytes.">Mitogen induction of ion channels in murine T lymphocytes.</a> J Gen Physiol. 89 (3), 405-420 (1987).</li><li>Nibbering, P. H., Zomerdijk, T. P., Tilburg, A. J., Furth, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mean+cell+volume+of+human+blood+leucocytes+and+resident+and+activated+murine+macrophages.">Mean cell volume of human blood leucocytes and resident and activated murine macrophages.</a> J Immunol Methods. 129 (1), 143-145 (1990).</li><li>Segel, G. B., Cokelet, G. R., Lichtman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+lymphocyte+volume:+importance+of+reference+particle+deformability+and+counting+solution+tonicity.">The measurement of lymphocyte volume: importance of reference particle deformability and counting solution tonicity.</a> Blood. 57 (5), 894-899 (1981).</li><li>Cooper, H. L., Braverman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+in+resting+and+growth-stimulated+human+peripheral+lymphocytes.+Evidence+for+regulation+by+a+non-messenger+RNA.">Protein synthesis in resting and growth-stimulated human peripheral lymphocytes. Evidence for regulation by a non-messenger RNA.</a> Exp Cell Res. 127 (2), 351-359 (1980).</li><li>Cooper, H. L., Braverman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close+correlation+between+initiator+methionyl-tRNA+level+and+rate+of+protein+synthesis+during+human+lymphocyte+growth+cycle.">Close correlation between initiator methionyl-tRNA level and rate of protein synthesis during human lymphocyte growth cycle.</a> J Biol Chem. 256 (14), 7461-7467 (1981).</li><li>Teague, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+changes+the+spectrum+but+not+the+diversity+of+genes+expressed+by+T+cells.">Activation changes the spectrum but not the diversity of genes expressed by T cells.</a> Proc Natl Acad Sci U S A. 96 (22), 12691-12696 (1999).</li><li>Tzur, A., Kafri, R., Lebleu, V. S., Lahav, G., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+growth+and+size+homeostasis+in+proliferating+animal+cells.">Cell growth and size homeostasis in proliferating animal cells.</a> Science. 325 (5937), 167-171 (2009).</li><li>Messele, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonradioactive+techniques+for+measurement+of+in+vitro+T-cell+proliferation:+alternatives+to+the+[3H]thymidine+incorporation+assay.">Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [3H]thymidine incorporation assay.</a> Clin Diagn Lab Immunol. 7 (4), 687-692 (2000).</li><li>Maghni, K., Nicolescu, O. M., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+cell+metabolic+colorimetric+assays+for+assessment+of+CD4++T+cell+proliferation:+comparison+to+5-bromo-2-deoxyuridine+(BrdU)+ELISA.">Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation: comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA.</a> J Immunol Methods. 223 (2), 185-194 (1999).</li><li>Weichert, H., Blechschmidt, I., Schr&#246;der, S., Ambrosius, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MTT-assay+as+a+rapid+test+for+cell+proliferation+and+cell+killing:+application+to+human+peripheral+blood+lymphocytes+(PBL).">The MTT-assay as a rapid test for cell proliferation and cell killing: application to human peripheral blood lymphocytes (PBL).</a> Allerg Immunol (Leipz). 37 (3-4), 139-144 (1991).</li><li>Huang, K. T., Chen, Y. H., Walker, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inaccuracies+in+MTS+assays:+major+distorting+effects+of+medium,+serum+albumin,+and+fatty+acids.">Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids.</a> Biotechniques. 37 (3), 410-412 (2004).</li><li>Rampersad, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+applications+of+Alamar+Blue+as+an+indicator+of+metabolic+function+and+cellular+health+in+cell+viability+bioassays.">Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays.</a> Sensors (Basel). 12 (9), 12347-12360 (2012).</li><li>Quent, V. M., Loessner, D., Friis, T., Reichert, J. C., Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrepancies+between+metabolic+activity+and+DNA+content+as+tool+to+assess+cell+proliferation+in+cancer+research.">Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research.</a> J Cell Mol Med. 14 (4), 1003-1013 (2010).</li><li>Augustine, N. H., Pasi, B. M., Hill, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+ATP+production+in+whole+blood+and+lymphocyte+proliferation+in+response+to+phytohemagglutinin.">Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin.</a> J Clin Lab Anal. 21 (5), 265-270 (2007).</li><li>Sottong, P. R., Rosebrock, J. A., Britz, J. A., Kramer, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+T-lymphocyte+responses+in+whole-blood+cultures+using+newly+synthesized+DNA+and+ATP.">Measurement of T-lymphocyte responses in whole-blood cultures using newly synthesized DNA and ATP.</a> Clin Diagn Lab Immunol. 7 (2), 307-311 (2000).</li><li>Wallace, P. K., Muirhead, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+2007:+a+proliferation+of+probes+and+applications.">Cell tracking 2007: a proliferation of probes and applications.</a> Immunol Invest. 36 (5-6), 527-561 (2007).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat Protoc. 2 (9), 2049-2056 (2007).</li><li>Teague, T. K., Munn, L., Zygourakis, K., Mcintyre, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+lymphocyte+activation+and+proliferation+by+video+microscopy+and+digital+imaging.">Analysis of lymphocyte activation and proliferation by video microscopy and digital imaging.</a> Cytometry. 14 (7), 772-782 (1993).</li><li>B&#246;hmer, R. M., Bandala-Sanchez, E., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+light+scatter+is+a+simple+measure+of+T-cell+activation+and+proliferation+but+is+not+universally+suited+for+doublet+discrimination.">Forward light scatter is a simple measure of T-cell activation and proliferation but is not universally suited for doublet discrimination.</a> Cytometry A. 79 (8), 646-652 (2011).</li><li>Lee, J., Sadelain, M., Brentjens, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+murine+primary+T+lymphocytes.">Retroviral transduction of murine primary T lymphocytes.</a> Methods Mol Biol. 506, 83-96 (2009).</li><li>Gunzer, M., Weishaupt, C., Planelles, L., Grabbe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+negative+enrichment+of+CD4++and+CD8++T+cells+from+murine+spleen+via+nylon+wool+adherence+and+an+optimized+antibody+cocktail.">Two-step negative enrichment of CD4+ and CD8+ T cells from murine spleen via nylon wool adherence and an optimized antibody cocktail.</a> J Immunol Methods. 258 (1-2), 55-63 (2001).</li><li>Trickett, A., Kwan, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+stimulation+and+expansion+using+anti-CD3/CD28+beads.">T cell stimulation and expansion using anti-CD3/CD28 beads.</a> J Immunol Methods. 275 (1-2), 251-255 (2003).</li><li>P&#232;ne, J., Rahmoun, M., Temmerman, S., Yssel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+anti-CD3/CD28+mAb+coupled+magnetic+beads+permitting+subsequent+phenotypic+analysis+of+activated+human+T+cells+by+indirect+immunofluorescence.">Use of anti-CD3/CD28 mAb coupled magnetic beads permitting subsequent phenotypic analysis of activated human T cells by indirect immunofluorescence.</a> J Immunol Methods. 283 (1-2), 59-66 (2003).</li><li>Sigal, N. H., Dumont, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin+A,+FK-506,+and+rapamycin:+pharmacologic+probes+of+lymphocyte+signal+transduction.">Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction.</a> Annu Rev Immunol. 10 (1), 519-560 (1992).</li><li>Fruman, D. A., Klee, C. B., Bierer, B. E., Burakoff, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+phosphatase+activity+in+T+lymphocytes+is+inhibited+by+FK+506+and+cyclosporin+A.">Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A.</a> Proc Natl Acad Sci U S A. 89 (9), 3686-3690 (1992).</li><li>Pollizzi, K. N., Waickman, A. T., Patel, C. H., Sun, I. H., Powell, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+size+as+a+means+of+tracking+mTOR+activity+and+cell+fate+of+CD4++T+Cells+upon+antigen+recognition.">Cellular size as a means of tracking mTOR activity and cell fate of CD4+ T Cells upon antigen recognition.</a> PLoS One. 10 (4), e0121710 (2015).</li><li>Pollizzi, K. N., Powell, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cells+by+mTOR:+the+known+knowns+and+the+known+unknowns.">Regulation of T cells by mTOR: the known knowns and the known unknowns.</a> Trends Immunol. 36 (1), 13-20 (2015).</li><li>Mandala, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+lymphocyte+trafficking+by+sphingosine-1-phosphate+receptor+agonists.">Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.</a> Science. 296 (5566), 346-349 (2002).</li><li>Qin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+and+FTY720+are+potent+inhibitors+of+the+transient+receptor+potential+melastatin+7+(TRPM7)+channels.">Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels.</a> Br J Pharmacol. 168 (6), 1294-1312 (2013).</li><li>Wulff, H., Kolski-Andreaco, A., Sankaranarayanan, A., Sabatier, J. M., Shakkottai, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulators+of+small-+and+intermediate-conductance+calcium-activated+potassium+channels+and+their+therapeutic+indications.">Modulators of small- and intermediate-conductance calcium-activated potassium channels and their therapeutic indications.</a> Curr Med Chem. 14 (13), 1437-1457 (2007).</li><li>Petho, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-proliferative+effect+of+cation+channel+blockers+in+T+lymphocytes+depends+on+the+strength+of+mitogenic+stimulation.">The anti-proliferative effect of cation channel blockers in T lymphocytes depends on the strength of mitogenic stimulation.</a> Immunol Lett. 171, 60-69 (2016).</li><li>Wulff, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+potent+and+selective+inhibitor+of+the+intermediate-conductance+Ca2+-activated+K++channel,+IKCa1:+a+potential+immunosuppressant.">Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant.</a> Proc Natl Acad Sci USA. 97 (14), 8151-8156 (2000).</li><li>Lang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+cell+volume+regulatory+mechanisms.">Functional significance of cell volume regulatory mechanisms.</a> Physiol Rev. 78 (1), 247-306 (1998).</li><li>Hue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+liver+carbohydrate+and+fatty+acid+metabolism+by+cell+volume.">Control of liver carbohydrate and fatty acid metabolism by cell volume.</a> Biochem Soc Trans. 22 (2), 505-508 (1994).</li><li>O'Flaherty, J. T., Kreutzer, D. L., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+aggregation+and+swelling+induced+by+chemotactic+agents.">Neutrophil aggregation and swelling induced by chemotactic agents.</a> J Immunol. 119 (1), 232-239 (1977).</li><li>Hsu, L. S., Becker, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+changes+induced+in+rabbit+polymorphonuclear+leukocytes+by+chemotactic+factor+and+cytochalasin+B.">Volume changes induced in rabbit polymorphonuclear leukocytes by chemotactic factor and cytochalasin B.</a> Am J Pathol. 81 (1), 1-14 (1975).</li><li>Rosengren, S., Henson, P. M., Worthen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration-associated+volume+changes+in+neutrophils+facilitate+the+migratory+process+in+vitro.">Migration-associated volume changes in neutrophils facilitate the migratory process in vitro.</a> Am J Physiol. 267, C1623-C1632 (1994).</li></ol>

The authors have no competing financial interests.

Here, we describe a technique for the rapid detection and quantification of T-cell blastogenic transformation using an automated cell counter. Under our conditions (250 ng/ml PMA and 250 nM ionomycin stimulation), the cell surface area was increased two-fold and the volume three-fold after 48 hr of activation. The assay is sufficiently sensitive to detect blasting during the first 12 hr of activation, where the cell volume increased only 1.25-fold compared to resting (Figure 2D). A more physiologically relevant mechanism of T-cell activation using anti-CD3 and anti-CD28 antibody-coated magnetic beads produced a significant blastogenic response, with an average 2.3-fold increase in volume (72 hr after activation, Figure 3D). Human mononuclear cells showed a 2.6-fold change in volume upon PMA/ionomycin stimulation for 48 hr (Figure 4). The ICR mouse splenic T cell average diameter and volume were determined to be 6.9 &#181;m and 1.9 x 10-4 nl, respectively. Human PBMCs (from one healthy donor) had an average diameter of 7.7 &#181;m and an average volume of 2.7 x 10-4 nl. Using this protocol, we also tested increasing concentrations of PMA for their ability to activate T cells. These single-cell results were consistent with proliferation assays performed at similar PMA concentrations.
Several compounds reported to affect cell proliferation were tested: FTY72031,32; the immunosuppressants cyclosporine A, FK506, and rapamycin27,28; and TRAM-34, a blocker of calcium-activated KCa3.1 channels33,35. We found that, at the tested concentrations, the most potent inhibitors of blastogenesis were CsA and FK506. Rapamycin had a smaller but statistically significant suppressive effect on blastogenesis. TRAM-34, on the other hand, did not affect blastogenesis.
CsA suppressed both blastogenesis and proliferation, but not completely (Figure 2B and 2C), demonstrating that automated cell counter measurement is a good correlate of drug efficiency in T-cell proliferation. In the presence of rapamycin together with CsA, blastogenesis was completely inhibited, suggesting that NFAT- and mTOR-mediated pathways in combination can fully account for T-cell enlargement upon mitogenic stimulation with PMA/ionomycin (Figure 3C).
The dynamic range of some proliferation assays is limited (see Figure 2). Proliferation assays, such as the one we have used (Figure 2C), report a signal that includes contributions from apoptotic and necrotic cells. Automated diameter change measurements do not have that limitation, as the measurements pertain to viable cells only. Cell viability is assessed by the exclusion of trypan blue stain from healthy, viable cells. In size measurements, using forward light scatter in flow cytometry doublet cell discrimination can be problematic22. In the automated cell counter measurements, no doublet population was found (Figure 1). Also, the measurement was sufficiently precise to distinguish between the effects of 100 and 200 nM cyclosporine (Table 1) and to detect blastogenesis within 12 hr of mitogenic stimulation (Figure 2D).
This new assay is particularly useful for small numbers of samples. Up to 15 samples can be measured in 1 hr. However, the assay has its limitations, most of which can be mitigated. Even though it can effectively resolve small (&#60;1 &#181;m) difference in cell diameter, the machine model we used has a lower detection threshold of 5 &#181;m. This limit can cause the overestimation of very small cell sizes, since it effectively excludes all cells with smaller diameters. However, newer models of the cell counter have detection thresholds of ~2 &#181;m, which should alleviate this problem. If there is too much debris in the sample, the instrument treats them as viable cells. Additionally, air bubbles can occasionally get into the flow cell and cause distortion of the cell images captured by the machine. The distortion does not appear to affect the viability determination, but it can affect the measured diameters. Therefore, it is recommended that all images be checked by the experimenter for air bubbles before the data from each trial are accepted for analysis. Since the software only draws circles around the cells, diameters of non-spherical cells may be skewed towards the long axis, making the assay less suitable for non-spherical cells.
This assay is quick, taking approximately 4 min per sample, compared to proliferation assays, which require a few hours. Data collection is also rather simple using the software included with the device. The software can export measurement data to a spreadsheet for analysis. Finally, it is a single-cell, as opposed to a population, measurement; both blastogenesis and proliferation are measured; and it distinguishes between viable and dead cells simultaneously. It can be used to evaluate various mouse strains for their ability to mount an immune response. The assay can also be used successfully for the measurement of blastogenesis in T cells from human donors (Figure 4), and it can also be used in other cell types.
Cell volume increases are known to contribute to the regulation of metabolism: cell swelling stimulates glutamine-induced glycogen synthesis and lipogenesis in hepatocytes36,37. Neutrophils display migration-associated volume increases of 35-60%, while chemotactic agents induce 10-15% swelling38-40. The current assay can potentially be used to study these processes.

T lymphocytes are the primary cells responsible for adaptive immunity in mammals. It is known that they respond to specific antigenic peptides presented by MHC molecules on the surface of antigen-presenting cells. Upon activation of a cognate T-cell receptor (TCR), the cell enlarges in a process termed blastogenic transformation, or blastogenesis. This process is detectable in the first ~6 hr after the stimulus is applied1. During blastogenesis, the volumes of individual T cells increase 2- to 4-fold2-6. Lymphocytes begin to proliferate in a process called clonal expansion, the purpose of which is to generate as many clones of the antigen-specific TCR-bearing cells as possible. The progeny cells then exert their immunologic function by differentiating into cytotoxic (CD8+) or helper (CD4+) effector T lymphocytes. Thus, na&#239;ve T lymphocytes in human or mouse blood are in the G0 (resting) phase of the cell cycle and support minimal metabolic activity. Upon exposure to antigens or mitogens, T cells reenter the cell cycle, with a concomitant stimulation of transcription and protein synthesis7-10. Mitogens, such as phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulate the lymphocytes through the activation of protein kinase C (PKC) and Ca2+-dependent signaling pathways1. The activation of T cells with PMA/ionomycin bypasses the TCR signaling steps.
In vitro proliferation assays are widely used for the purpose of evaluating lymphocyte function and response to stimuli. Proliferation readings are typically taken one to three days after the start of T-cell stimulation and reflect the collective state of hundreds or thousands of cells. The potency of various mitogens and immunomodulatory drugs in vitro can be evaluated by simply measuring proliferation rates in the presence of these compounds. Some of these assays and their limitations are discussed below.
For direct cell number counting, the procedure is time consuming, with a high probability of operator errors.
For DNA synthesis, the 3H-thymidine incorporation assay measures DNA synthesis, but its major limitation is its radiotoxicity. A non-radioactive alternative is BrdU, but the range of linear response for the cell growth is limited, and antibody treatment is required, which increases the number of steps in the procedure11,12.
For metabolic activity, tetrazolium salts (MTT, MTS, XTT, and WST-1) and resazurin dye-based colorimetric assays report the general metabolic state of dividing cell populations. However, MTT is not soluble in the culture medium, requiring additional wash steps, thus incorporating errors in the measurement; XTT needs additional components to reduce efficiently; MTS-, WST-1-, and resazurin-based measurements are affected by the culture medium pH and its components serum, albumin or phenol red13-16. These assays do not measure the actual number of viable cells but rather estimate the combined enzyme activities. Therefore, the proliferation rate may not be accurately determined by metabolic assays because of the non-linear correlation between cell number and dye reduction12,17.
For measuring ATP concentration, T-cell activation-induced increases in ATP correlate with proliferation. However, elevation of intracellular ATP is one of the initial steps of T cell activation; many steps behind is the actual proliferation17,18.
For dye dilution assay, CFSE fluorescent dye stains cells by covalently binding to intracellular proteins. The dye shows a proliferation-dependent decrease in fluorescent intensity, which can track the number of cell divisions. However, because of covalent protein labeling, the functions of these proteins can be compromised. The dye is toxic to the cells at higher concentrations. At lower dye concentrations, however, the initial fluorescence intensity is reduced, decreasing the number of cell divisions that can be tracked. Additionally, after labeling with CFSE, there is a proliferation-independent ~50% loss of initial fluorescence during the first 24 to 48 hr period, which limits the dynamic range of this assay19,20.
Most of these assays reflect the collective state of large numbers of cells and require the treatment of the cells with fluorescent dyes. Necrotic and apoptotic cells might also contribute to these measurements, unless they are removed from the analysis by staining with chemicals or antibodies.
Lymphocyte blastogenesis can be evaluated by a variety of methods, such as optical microscopy or flow cytometry4,21,22. Here, we describe a rapid method for the measurement of T-cell sizes using an automated cell counter, which collects real-time cell images that are stored and can be re-analyzed at a later time. In addition to size measurements, this device provides precise cell numbers and the percentage of viable cells, as determined by trypan blue stain exclusion. The device used in this protocol is commercially available, and the manufacturer tested the precision of the instrument using three different instruments and several concentration and viability controls. Results of these studies demonstrated a coefficient of variance that was generally below 6%. As noted in the protocol, the device is calibrated on a regular basis with 6 &#181;m and 8 &#181;m diameter polystyrene beads. The advantages of using a cell counter to differentiate between resting T cells and T lymphoblasts based on cell diameter is the ease of use and the automated nature of the analysis. The software is capable of drawing a circle around each cell and calculating the cell diameter. Additionally, the images are visible to the operator, who can verify the accuracy of the instrument in identifying the cells and correctly drawing a circle around them. In terms of limitations, the instrument cannot per se differentiate between debris and cells; therefore, it is important that the operator views every image as it is being processed. There is a potential for incorporating air bubbles, which will decrease the number of usable fields for analysis; however, this is rare if the regular flushing maintenance is performed.
In this study, groups of splenic T lymphocytes were stimulated with ionomycin and increasing concentrations of PMA for 12-48 hr. PMA concentrations as low as 2 ng/ml induced both a robust blastogenic response and significant proliferation. Measurements of the effects of several drugs, such as the immunosuppressants cyclosporine A (CsA), FK506 (tacrolimus), and rapamycin (sirolimus), as well as ion channel blockers TRAM-34 and FTY720 (fingolimod), on blastogenesis demonstrated good agreement with reported effects on proliferation. The blastogenic response of human PBMCs to PMA/ionomycin and murine T-cell stimulation with anti-CD3 and anti-CD28 antibody-coated magnetic beads were also measured.
The cell counter assay quantifies both blastogenesis and the proliferation rate (cell density) simultaneously but separately, unlike the abovementioned methods, which look at a combination of these effects. The presented protocol provides a rapid and robust technique for evaluating the potency of mitogenic and immunomodulatory agents.

<table border="0" cellpadding="0" cellspacing="0" width="1149">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">RPMI-1640&#160;</td>
			<td style="width:283px;">Lonza</td>
			<td style="width:216px;">BW12-702F</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 &#956;m nylon cell strainers&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nylon wool fiber columns</td>
			<td>Polysciences, Inc.&#160;</td>
			<td>21759-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml conical tubes&#160;</td>
			<td>The Lab Depot</td>
			<td>TLD431697</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well cell culture treated plates</td>
			<td>USA Scientific</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well cell culture treated plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>130188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher BioReagents</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTiter 96 AQueous One Solution Cell Proliferation Assay&#160;</td>
			<td>Promega</td>
			<td>G3582</td>
			<td>MTS based assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclosporine A</td>
			<td>Sigma-Aldrich</td>
			<td>30024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FK506</td>
			<td>Cayman Chemical Company</td>
			<td>104987-11-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapamycin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-3504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRAM-34</td>
			<td>Sigma-Aldrich</td>
			<td>T6700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FTY720</td>
			<td>Sigma-Aldrich</td>
			<td>SML0700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads Mouse T-Activator CD3/CD28</td>
			<td>Gibco</td>
			<td>11456D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phorbol 12-myristate 13-acetate (PMA)&#160;</td>
			<td>Acros Organics&#160;</td>
			<td>356150010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ionomycin calcium salt</td>
			<td>Sigma-Aldrich</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>MP Biomedicals&#160;</td>
			<td>ICN1670049</td>
			<td>100 x stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline (DPBS)&#160;</td>
			<td>HyClone</td>
			<td>SH30378.02</td>
			<td>10 x stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,4-dithiothreitol (DTT)&#160;</td>
			<td>Research Products International</td>
			<td>12/3/3483</td>
			<td>reducing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8 &#181;m micro particle size standard&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>84192-5ML-F</td>
			<td>Actual&#160; 8.02 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 &#181;m micro particle size standard&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>89756-5ML-F</td>
			<td>Actual 6.084 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single magnetic separation stand for 1.5 - 2 mL tube</td>
			<td>V&#38;P Scientific, Inc.</td>
			<td>VP772F5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture incubator</td>
			<td>Forma Scientific&#160;</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy H1 hybrid reader&#160;</td>
			<td>Bio Tek</td>
			<td>BTH1M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vi-CELL cell viability analyzer&#160;</td>
			<td>Beckman Coulter</td>
			<td>731050</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid quantification of mitogen-induced blastogenesis in t lymphocytes for identifying immunomodulatory drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

We used this assay to compare lymphocyte blast formation during stimulation with PMA, a phorbol ester, and ionomycin, a calcium ionophore. Figure 1A shows the frequency distribution of diameters of resting and pharmacologically activated splenic T cells. Treatment of cells with PMA/ionomycin for ~2 days resulted in a significant shift in the median of the distribution towards bigger diameters (see, for example reference4). The number of cells with smaller diameters was accordingly reduced. In order to ascertain that our device reported the correct diameters, we performed two calibrations with polystyrene beads of 6 &#181;m and 8 &#181;m diameters (see Materials Table). Figures 1B and 1C show readings from activated T cells superimposed with an 8 &#181;m standard and resting T cells superimposed with a 6 &#181;m standard. Figure 1D shows the control bead sizes reported by the manufacturer plotted against the median diameters measured with our automated cell counter. It appeared that the 6 &#181;m size was slightly overestimated in our measurements, probably due to the measurement threshold of the instrument having a lower limit of 5 &#181;m. However, the standard deviation of the bead diameters calculated from the cell counter measurements was in agreement with the standard deviation reported by the manufacturer. We conclude from these experiments that this device can be used reliably to compare resting and mitogen-stimulated mouse T-cell sizes and that the device can accurately measure the actual cell diameters.
We then proceeded to test the dependence of the mean diameter increase on PMA concentration and found that, at a fixed ionomycin concentration of 250 nM, the PMA effect was not significantly changed by elevating its concentration from 2 to 250 ng/ml (Figure 2A). Using the MTS-based assay, we measured T-cell proliferation at 50 and 250 ng/ml PMA and found no appreciable difference (Figure 2C), in agreement with our cell diameter data. The calcineurin inhibitor drugs cyclosporine A (300 nM) and FK506 (1 nM) suppressed both blastogenesis (Figure 2B) and proliferation (Figure 2C). Inhibition was not complete in both cases, however. Measurements performed 12 hr after PMA/ionomycin addition showed a 7.2% and 6.8% increase in diameter for 50 ng/ml and 250 ng/ml PMA, respectively (Figure 2D). Size increases at these two concentrations were not significantly different, as was the case for 48 hr stimulation (compare to Figure 2A).
The cell diameter data collected from resting and activated T cells after 48 hr of PMA/ionomycin stimulation are summarized in Table 1. The mean surface area and volume of resting ICR mouse splenic T cells were 151.4 &#181;m2 and 1.9 x 10-4 nl, respectively. The mean surface area and volume of activated T cells were 300.6 &#181;m2 and 5.9 x 10-4 nl, respectively. The overall average diameter increase for all activated cells was 40.92% (Table 1).
We also tested other chemical compounds reported to be immunosuppressive: CsA, FK506, rapamycin, FTY720, and TRAM-34. In Figure 3, cell size measurements obtained in resting and activated T cells in the absence and presence of these drugs are shown. CsA and FK506, which target the calcineurin-dependent nuclear factor of activated T cells (NFAT)27,28, were the most potent, inhibiting the blastogenic response by nearly 72% (Figure 3A and 3B). Interestingly, with increased PMA concentrations, the potency of CsA and FK506 dropped slightly, even though there was no additional cell diameter increase in the absence of these drugs (Figure 2A and 2B). Rapamycin, an immunosuppressant that inhibits mammalian target of rapamycin (mTOR)29,30, had a moderate but statistically significant effect (Figure 3A, 3B and 3C). FTY720 is a sphingosine 1-phosphate receptor agonist that inhibits lymphocyte egress into circulation31 and was recently found to inhibit TRPM7, a cation channel highly expressed in T lymphocytes32. Both FTY720 and rapamycin had a comparable effect on blastogenesis, reducing it from the average of a 52% increase in diameter to approximately 34% (Figure 3A and 3B). TRAM-34 is a blocker of the calcium-activated potassium channel KCa3.133. Tested at 700 nM, TRAM-34 was ineffective in murine T cells, in accordance with a recent human T-cell proliferation study; its potency may depend on the nature and strength of mitogenic stimulation34 (Figure 3A and 3B). 700 nM TRAM-34 was effective in blocking KCa3.1 channels in our patch clamp electrophysiology experiments (data not shown). This comparison was made between resting and drug-exposed cells in multiple trials within the same experiment at 48 hr. When rapamycin and CsA were used together, blastogenesis was completely inhibited (Figure 3C).
Activation of murine T cells with anti-CD3/anti-CD28-coated magnetic beads for 72 hr produced a 27% increase in the average diameter, which was reduced to 5% in the presence of CsA (Figure 3D). Human mononuclear cells upon PMA/ionomycin activation for 48 hr increased in diameter by 33%, and activation in the presence of CsA decreased the response to 23% (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/55212/55212fig1.jpg" /> 
Figure 1: Frequency distributions of murine splenic T-cell diameters. (A) Black columns indicate resting and red columns indicate PMA/ionomycin-activated (48 hr) cells. (B) Distributions of activated splenic T cells superimposed on an 8 &#181;m particle size calibration standard. (C) Distributions of resting T cells superimposed on a 6 &#181;m calibration standard. The numbers of cells and beads used are indicated in boxes. (D) Median diameter measured by the cell counter plotted against the bead diameter reported by the manufacturer. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55212/55212fig2.jpg" /> 
Figure 2: Dependence of murine T-cell activation on PMA concentration. (A) Mean diameters of T cells either untreated or treated with 2, 50, and 250 ng/ml PMA at a fixed ionomycin concentration (250 nM). (B) Cell counter measurements of resting and activated (2, 50, 250 ng/ml PMA) T cells in the absence and presence of 300 nM CsA or 1 nM FK506. (C) MTS proliferation assay at 50 and 250 ng/ml PMA with 250 nM ionomycin in the absence and presence of 300 nM CsA. Significant differences (p &#60; 0.001) are indicated with asterisks. n is the total number of trials. Data are from cells isolated from 3 mice. (D) Cell diameters of resting and activated T cells (250 ng/ml PMA and 250 nM ionomycin) 12 hr after activation in the absence and presence of 300 nM CsA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55212/55212fig3.jpg" /> 
Figure 3: Effects of CsA, FK506, FTY720, rapamycin, and TRAM-34 on cell size. (A and C) Average diameters of resting and PMA/ionomycin-activated murine T cells in the absence and presence of drugs. Concentrations are shown in the box. (B) Data from A expressed as the percent increase over the resting T-cell diameter. Lymphocyte activation was performed with 250 ng/ml PMA and 250 nM ionomycin, and the measurements were taken after 48 hr. (D) Cell counter measurements of resting and anti-CD3/CD28-activated T cells, 72 hr after activation in the absence and presence of 300 nM CsA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55212/55212fig4.jpg" /> 
Figure 4: Blastogenesis in human PBMCs upon mitogen stimulation. (A) Black columns indicate resting and red columns indicate activated PBMCs. (B) Mean diameters of resting and activated PBMCs in the absence and presence of 300 nM CsA. Cells were activated with 250 ng/ml PMA and 250 nM ionomycin, and the measurements were taken after 48 hr of activation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Column1</td>
			<td>Resting</td>
			<td>Activated</td>
			<td>CsA 100 nM</td>
			<td>CsA 200 nM</td>
		</tr>
		<tr>
			<td>Average Diameter</td>
			<td>6.94 &#181;m</td>
			<td>9.78 &#181;m</td>
			<td>8.19 &#181;m</td>
			<td>7.92 &#181;m</td>
		</tr>
		<tr>
			<td>Average Increase in Diameter</td>
			<td></td>
			<td>40.92%</td>
			<td>17.88%</td>
			<td>14.09%</td>
		</tr>
		<tr>
			<td>Average Surface Area</td>
			<td>151.37 &#181;m&#178;</td>
			<td>300.61 &#181;m&#178;</td>
			<td>210.56 &#181;m&#178;</td>
			<td>197.22 &#181;m&#178;</td>
		</tr>
		<tr>
			<td>Average Increase in Surface Area</td>
			<td></td>
			<td>98.59%</td>
			<td>39.00%</td>
			<td>30.16%</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of the average cell diameter and relative increases in surface area. Measurements were performed 48 hr after activation with 250 ng/ml PMA and 250 nM ionomycin.

Watch this Scientific Journal Video about Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs at JoVE.com

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

rapid-quantification-mitogen-induced-blastogenesis-t-lymphocytes-for

Research

JoVE Journal

Immunology and Infection

18.7K Views.  Wright State University. T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters. 

Video: Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.