This work was supported by the Office of Tourism, Trade, and Economic Development of Florida.

1. Preparation of Effector CD8+ T Cells
<ol>
	<li>Thaw autologous cryopreserved PBMCs (2-3 vials of 50&#160;x 106 cells) by transferring the cryovial from liquid nitrogen to a 37 &#176;C water bath.</li>
	<li>Wash the cells by filling the tube to 50 ml with complete RPMI (4 mM L-glutamine, and 100 U/ml penicillin and streptomycin, supplemented with 10% FBS). Count PBMCs and resuspend cells at a concentration of 5 x 106/ml in complete RPMI.</li>
	<li>Add specific peptide (5 &#181;g/ml) and IL-2 (10 ng/ml) to PBMCs.</li>
	<li>Set-up culture in 96 deep well plate; seed 1 ml of cell suspension to each well.</li>
	<li>After 3 days of culture, replace half of the cell culture medium with fresh complete RPMI.</li>
	<li>After 6 days of culture, collect all PBMCs with multichannel pipette and transfer cells in sterile reservoir.</li>
	<li>Count, wash, and resuspend PBMCs at 5 x 107/ml in the recommended separation buffer in 14 ml round bottom tubes. Add human CD8+ T cell enrichment cocktail at 50 &#181;l/ml cells. Mix and incubate at room temperature for 10 min. Add magnetic particles at 150 &#181;l/ml and incubate for 5 min.</li>
	<li>Bring the cell suspension up to 7 ml by adding the separation buffer. Proceed to immunomagnetic isolation of untouched CD8+ T cell by placing the tube into the magnet. After 5 min, with the tube still in the magnet, pour the cells of interest into a new 15 ml conical tube. Take a small aliquot and stain the cells with antibodies against CD3 and CD8 in 1X PBS-2% FBS for 30 min at 4 &#176;C. The purity of the CD8+ T cells can then be measured via flow cytometry with an expected purity of 95% or higher.</li>
	<li>Resuspend CD8+ T cells in 450 &#181;l with complete RPMI.</li>
	<li>Add 225 &#181;l of complete RPMI into 5 screw cap tubes.</li>
	<li>Prepare serial dilutions (from 1:2 to 1:32) by transferring 225 &#181;l of CD8+ to the next tube (see Figure 1 upper panel).</li>
</ol>
2. Preparation of Target CD4+ T Cells
<ol>
	<li>Thaw autologous cryopreserved PBMCs (1 vial of 50 x 106 cells) by transferring the cryovial from liquid nitrogen to a 37 &#176;C water bath.</li>
	<li>Transfer the cell suspension to a 50 ml conical tube containing 10 ml of RPMI.</li>
	<li>Wash cells and resuspend PBMCs at 5 x 107/ml with separation buffer in 14 ml round bottom tubes. Add human CD4+ T cell enrichment cocktail at 50 &#181;l/ml cells. Mix and incubate at room temperature for 10 min. Add magnetic particles at 100 &#181;l/ml and incubate for 5 min at room temperature.</li>
	<li>Bring the cell suspension up to 7 ml by adding separation buffer. Proceed to the isolation of CD4+ T cells by placing the tube into the magnet. After 5 min, with the tube still within the magnet, pour the desired cells into a new 15 ml conical tube. Take a small aliquot and stain the cells with antibodies against CD3 and CD4 in 1x&#160;PBS-2% FBS for 30 min at 4 &#176;C. The purity of the CD4+ T cells can then be measured via flow cytometry with an expected purity of 95% or higher.</li>
	<li>Count CD4+ T cells, split them into two 15 ml conical tubes and wash in warm 1x&#160;PBS.</li>
	<li>Prepare a 0.4 &#181;M CFSE-high working solution in warm PBS.</li>
	<li>Prepare a 0.04 &#181;M CFSE-low working solution by diluting a portion of the 10x&#160;CFSE high working solution in warm PBS.</li>
	<li>Resuspend cells at 20 x 106/ml of PBS (if the number of CD4+ T cells is less than 10 x 106/ml resuspend cells in 0.5 ml of PBS) and stain half of the CD4+ T cells by adding one equal volume of 0.4 &#181;M CFSE-high working solution (final concentration 0.2 &#181;M). For example, for 1 ml of cells, add 1 ml of the CFSE working solution. Stain the remaining half of the CD4+ by adding one equal volume of 0.04 &#181;M CFSE-low working solution (final concentration 0.02 &#181;M). Stain cells for 15 min at 37 &#176;C in 5% CO2 atmosphere.</li>
	<li>After the incubation, pellet cells at 1,500 rpm for 5 min and resuspend in 1 ml of warm complete RPMI to quench the labeling reaction.</li>
	<li>Pulse CFSE-low CD4+ T cells by adding the peptide of interest at a final concentration of 5 &#181;g/ml in the complete RPMI and incubate for 45 min in 5% CO2 atmosphere at 37 &#176;C.</li>
	<li>Wash CFSE-high and CFSE-low CD4+ T cells in complete RPMI. Repeat for a total of two washes. Resuspend at a concentration of 2 x&#160;105 cells/ml each in complete RPMI.</li>
	<li>Mix the two target populations at a ratio of 1:1 (CFSE-High:CFSE-Low) (see Figure 1 lower panel).</li>
</ol>
3. Coculture
<ol>
	<li>From each dilution of CD8+ T cells, seed 100 &#181;l in duplicate to round-bottom 96-well plate.</li>
	<li>Add the mixed target CD4+ T cells (100 &#181;l) to each dilution of effector cells for a final volume of 200 &#181;l.</li>
	<li>To measure the basal apoptosis, seed 3 wells with targets cells alone.</li>
	<li>Incubate coculture for 6 hr in 5% CO2 atmosphere at 37 &#176;C.</li>
</ol>
4. Flow Cytometry Staining and Acquisition
<ol>
	<li>Transfer cells to a V-bottom 96-well plate and stain with PE-conjugated pMHC tetramers for 15 min in 5% CO2 atmosphere at 37 &#176;C.</li>
	<li>Wash in 1x&#160;PBS-2% FBS and stain cells with Far Red LIVE/DEAD fixable dead cell, &#945;CD3-Alexa700, &#945;CD8-Percp, &#945;CD4-Brilliant Violet 650 for 30 min at 4 &#176;C, protected from light.</li>
	<li>Wash cells once with 1x&#160;PBS-2% FBS and resuspend in 100 &#181;l with 2% formaldehyde in 1x&#160;PBS.</li>
	<li>Acquire cells on flow cytometer.</li>
</ol>

<ol>
	<li>Appay, V., Van Lier, R. A. W., Sallusto, F., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytometry. 73, 975-983 (2008).</li><li>Betts, M. R., Harari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+and+function+of+protective+T+cell+immune+responses+in+HIV.">Phenotype and function of protective T cell immune responses in HIV.</a> Curr. Opin. HIV AIDS. 3, 349-355 (2008).</li><li>Roder, J. C., 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=A+new+immunodeficiency+disorder+in+humans+involving+NK+cells.">A new immunodeficiency disorder in humans involving NK cells.</a> Nature. 284, 553-555 (1980).</li><li>Barber, D. L., Wherry, E. J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+rapid+in+vivo+killing+by+memory+CD8+T+cells.">Cutting edge: rapid in vivo killing by memory CD8 T cells.</a> J. Immunol. 171, 27-31 (2003).</li><li>Byers, A. M., Kemball, C. C., Moser, J. M., Lukacher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+rapid+in+vivo+CTL+activity+by+polyoma+virus-specific+effector+and+memory+CD8++T+cells.">Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells.</a> J. Immunol. 171, 17-21 (2003).</li><li>Devevre, E., Romero, P., Mahnke, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LiveCount+Assay:+concomitant+measurement+of+cytolytic+activity+and+phenotypic+characterisation+of+CD8(+)+T-cells+by+flow+cytometry.">LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry.</a> J. Immunol. Methods. 311, 31-46 (2006).</li><li>Altman, J. D., 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=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274, 94-96 (1996).</li><li>Mbitikon-Kobo, F. M., Bonneville, M., Sekaly, R. P., Trautmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+measurement+of+the+cytotoxic+capacity+of+human+primary+antigen-specific+CD8+T+cells.">Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells.</a> J. Immunol. Methods. 375, 252-257 (2012).</li><li>Bryant, J., Day, R., Whiteside, T. L., Herberman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+lytic+units+for+the+expression+of+cell-mediated+cytotoxicity.">Calculation of lytic units for the expression of cell-mediated cytotoxicity.</a> J. Immunol. Methods. 146, 91-103 (1992).</li><li>Varadarajan, N., 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=A+high-throughput+single-cell+analysis+of+human+CD8(+)+T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8(+) T cell functions reveals discordance for cytokine secretion and cytolysis.</a> J. Clin. Invest. 121, 4322-4331 (2011).</li><li>Trautmann, L., 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=Profound+metabolic,+functional,+and+cytolytic+differences+characterize+HIV-specific+CD8+T+cells+in+primary+and+chronic+HIV+infection.">Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection.</a> Blood. 120, 3466-3477 (2012).</li><li>Hildemann, S. 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=High+Efficiency+of+Antiviral+CD4(+)+Killer+T+Cells.">High Efficiency of Antiviral CD4(+) Killer T Cells.</a> PLoS One. 8, (2013).</li></ol>

The authors declare that they have no competing financial interests.

The assay described here allows for the quantification of the principal function of CD8+ T cells: the cytolytic activity. The accurate measurement of this function is of paramount importance when characterizing effector functions of CD8+ T cells, as previous studies reported discordance between this function and the cytokine secretion of antigen-specific CD8+ T cells10,11. Due to the short coculture incubation time (6 hr) and the use of autologous target cells, there is low rate of nonspecific killing of target cells. This assay is therefore highly specific and reliable. This assay can also be easily modified to analyze different cell subsets. The effector CD8+ T cells can be replaced by other cell types, such as CD4+ T cells as recently described in the in vivo killing assay12. The target CD4+ T cells can also be replaced by other cell types, as we previously described the use of autologous B cells as target cells8. In this system, the origin of the antigen can be replaced as well. For example, target cells could be infected with a virus instead of pulsed with peptides. This protocol provides an accurate method to quantify the cytolytic activity of antigen-specific CD8+ T cells. As this method is versatile and easy to perform, measuring the killing capacity of effector cells might be performed more often in the quantification of CD8+ T cell functions.

Cytolytic activity is the major function of CD8+ T cells but is still rarely measured as assays used for this purpose are cumbersome and have been difficult to standardize. Accurate measurement of this function is of paramount importance when characterizing effector functions of CD8+ T cells, as no reliable predictors of effective cell-mediated cytotoxicity have been described yet1,2. Here, we propose a new functional assay to measure the cytotoxic activity of antigen-specific CD8+ T cells on target CD4+ T cells. Several approaches have been developed to provide alternatives to the gold standard, chromium release assay. We present here a cell-based assay that reveals the entire process of killing as it measures the death of live target cells. This method was derived from protocols of flow cytometry-based cytotoxic assays in vivo in mice4,5 and in vitro in humans6. In this protocol, the antigen-specific CD8+ T cells contained in the total CD8+ T cell population are used as effector cells and autologous CD4+ T cells are used as target cells. Effector CD8+ T cells of interest are enumerated using MHCI/peptide tetramers7. Death of target cells is calculated by the ratio between peptide loaded/nonloaded CD4+ T cells. We have previously shown that this method was reproducible, sensitive, specific and did not depend on the number of effector cells within the total CD8+ T cell population8. By enumerating both the number of effector and target cells in the coculture assay, the intrinsic capacity of CD8+ T cells to kill target cells can be calculated and expressed in lytic units9.

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">EasySep Human CD4+ T Cell Enrichment Kit</td>
			<td style="width:132px;">Stem Cell</td>
			<td style="width:155px;">19052</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">EasySep Human CD8+ T Cell Enrichment Kit</td>
			<td>Stem Cell</td>
			<td>19053</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace CFSE Cell Proliferation Kit - For Flow Cytometry</td>
			<td>Invitrogen</td>
			<td>C34554</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Far Red LIVE/DEAD fixable dead cell</td>
			<td>Invitrogen</td>
			<td>L10120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 700 Mouse Anti-Human CD3</td>
			<td>BD</td>
			<td>557943</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 650 Anti-Human CD4 Antibody</td>
			<td>Biolegend</td>
			<td>317435</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP anti-human CD8 Antibody</td>
			<td>Biolegend</td>
			<td>344707</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium 1x</td>
			<td>VWR</td>
			<td>10-040-CV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin 100x Solution</td>
			<td>Invitrogen</td>
			<td>15070063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum - Gold, Heat Inactivated</td>
			<td>PAA</td>
			<td>A15-252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well plates; Treated; U-bottom; with lid</td>
			<td>BD Falcon</td>
			<td>353077</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 ml sterile polystyrene, individually wrapped reservoirs</td>
			<td>USA Scientific</td>
			<td>2321-2230</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well U-bottom, sterile 2 ml deep well plates</td>
			<td>Thermo Scientific</td>
			<td>275743</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human IL-2 (v126), research grade</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-096</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RoboSep Buffer, separation buffer</td>
			<td>Stem cell</td>
			<td>20104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Polypropylene Conical Tube</td>
			<td>BD Falcon</td>
			<td>352097</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 ml round bottom tube, polypropylene</td>
			<td>BD Falcon</td>
			<td>352059</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The Big Easy EasySep Magnet</td>
			<td>Stem cell</td>
			<td>18001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1x</td>
			<td>VWR</td>
			<td>21-040-CV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well; V-bottom plate with lid</td>
			<td>Fisher</td>
			<td>12-565-481</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution, ACS reagent, 37 wt % in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>252549-500ML</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml screw cap microtube, conical</td>
			<td>Sarstedt</td>
			<td>72.692.005</td>
		</tr>
	</tbody>
</table>

cell-based flow cytometry assay to measure cytotoxic activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

The schematic in Figure 1 summarizes the assay. Purified CD8+ T cells (effector cells) were resuspended in 450 &#181;l of complete RPMI and serial dilutions were performed by adding 225 &#181;l of CD8+ T cells to the next tube containing 225 &#181;l of media (upper panel). Purified CD4+ T cells (target cells) were counted, split into two tubes and stained with two different concentrations of CFSE (high and low) as described in the protocol. CFSE-low CD4+ T cells were pulsed with the peptide of interest and CFSE-high CD4+ T cells remained unloaded as a control. CFSE-high and CSFE-low CD4+ T cells were resuspended at 2 x&#160;105/ml and mixed at a ratio of 1:1 (lower panel). The mixed CD4+ T cells were seeded at 100 &#181;l in each well. Three wells containing the mixed CD4+ T cells were used for triplicate of CD4+ T cells alone (wells in red). 100 &#181;l of each CD8+ T cell dilution was added in duplicate to the remaining wells containing the mixed CD4+ T cells (wells in green). The killing of target cells was assessed by flow cytometry after 6 hr of coculture.
After incubation, cells were analyzed and gated as in Figure 2. The ratio of viable CFSE-high versus CFSE-low target cells, un-pulsed or peptide pulsed was analyzed from a small FSC-A/SSC-A gate. Live cells were gated on LIVE/DEAD negative. CD4+ T cells were plotted on CFSE in order to analyze the ratio CFSE-low versus CFSE-high cells. A larger FSC-A/SSC-A gate was then created to enumerate the total number of effector and target cells after 6 hr&#160;incubation, either dead or alive. Tetramer+ CD8+ T cells and CFSE-low CD4+ T cells were plotted in order to analyze the ratio effector versus target cells. The total number of effector CD8+ T cells was determined by gating on CD8+ and tetramer+ cells. The total number of target cells was determined by gating on CD4+ T cells CFSE-low and LIVE/DEAD positive and negative cells.
The results presented in Figure 3 show that the ratio of CFSE-low versus CFSE-high cells (target ratio) was 1:1 when CD4+ target cells were cultured in absence of effector cells. However, the ratio of CFSE-low versus CFSE-high cells was drastically decreased when CFSE-low cells pulsed with peptide were incubated with the total CD8+ T cell population containing antigen-specific CD8+ effector T cells. As the ratio decreased, the CFSE-low CD4+ pulsed target cells became LIVE/DEAD positive. The dilution of the effector cells (right panel) resulted in restoration of the CFSE ratio and decreased the number of LIVE/DEAD positive peptide-pulsed CFSE-low CD4+ target T cells.
For each well, the ratio between pulsed and un-pulsed live CD4+ target T cells (CFSE-low/CFSE-high) in the presence of different dilutions of effector CD8+ T cells was measured by calculating the ratio between antigen-pulsed CFSE-low CD4+ T cells and un-pulsed CFSE-high CD4+ T cells after 6 hr of culture with effector CD8+ T cells. The percentage of specific lysis for each well was calculated as follows: % specific lysis = 100 - ((CFSE low/CFSE high) in the presence of effector cells / mean of the three wells containing target cells alone in the absence of effector cells) x100. To determine the intrinsic cytolytic capacity of antigen-specific CD8+ T cells and compare results between donors, the cytolytic activity was expressed in lytic units as previously reported8. The effector versus target ratio (E/T) was measured by calculating the ratio between total tetramer+ CD8+ T cells and total CFSE-low CD4+ T cells for each well. The percentage of specific lysis was plotted in function of the E/T ratio in a log scale as illustrated in Figure 4. A linear regression was calculated from the plot and the equation of the trendline was used to calculate the lytic units LU30/106 cells (i.e.&#160;the number of CD8+ effectors T cells required to kill 30% of 106 target CD4+ T cells).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51105/51105fig1highres.jpg" src="/files/ftp_upload/51105/51105fig1.jpg" width="600px" /> 
Figure 1. Schematic representation of the cytotoxic assay coculture. Purified CD8+ T cells were resuspended in 450 &#181;l of complete RPMI and 5 serial dilutions were performed. Purified CD4+ T cells were counted, split into two tubes and stained with two concentrations of CFSE (0.02 &#181;M CFSE-low and 0.2 &#181;M CFSE-high). Only CFSE low CD4+ T cells were pulsed with 5 &#181;g of specific peptide. After 45 min, both CFSE low and high CD4+ T cells were washed 2x, resuspended at 2 x 105/ml and mixed at a ratio of 1:1. 100 &#181;l of mixed CD4+ were seeded in a 96-well plate in the presence or absence of 100 &#181;l of each CD8+ T cell dilution for 6 hr. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51105/51105fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51105/51105fig2highres.jpg" src="/files/ftp_upload/51105/51105fig2.jpg" width="600px" /> 
Figure 2. Gating strategy of the cytotoxic assay. From a smaller FSC-A/SSC-A gate, the ratio of CFSE-low versus CFSE-high cells was analyzed within the CD4+ LIVE/DEAD negative cells. From a larger FSC-A/SSC-A gate, effector cells were analyzed by gating on CD8+ T cells and tetramer+ cells. The number of target cells was analyzed by gating on CD4+ T cells, CFSE-low and LIVE/DEAD positive and negative cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51105/51105fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/51105/51105fig3highres.jpg" src="/files/ftp_upload/51105/51105fig3.jpg" width="500px" /> 
Figure 3. Representative results of FACS staining. CFSE-low/CFSE-high ratio (left panels), target cells (middle panels) and effector cells (right panels) in the different coculture conditions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51105/51105fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/51105/51105fig4highres.jpg" src="/files/ftp_upload/51105/51105fig4.jpg" width="600px" /> 
Figure 4. Linear regression of specific lysis in function of the effector/target ratio. The equation of the trendline is used to calculate the lytic units LU30/106 cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51105/51105fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity at JoVE.com

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

cell-based-flow-cytometry-assay-to-measure-cytotoxic-activity

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.9K Aufrufe.  Vaccine and Gene Therapy Institute of Florida. This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Serie: Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Forschung

Immunologie und Infektion

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Mensch In Vitro Suppression als Screening-Tool für die Anerkennung von Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Bestimmung der optimalen zytotoxische Aktivität der Menschenrechte Her2neu spezifischen CD8 T-Zellen durch Vergleich der Cr51 Freisetzungsassay dem xCELLigence-System

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

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.

Eine einfache und schnelle Protokoll zum Nicht enzymatisch Dissociate Frische menschlichen Geweben zur Analyse der infiltrierenden Lymphozyten

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

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Durchflusszytometrie-basierte Assay zur Überwachung von NK-Zell-Funktionen

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluoreszenz-aktivierte Zellsortierung zur Reinigung von Plasmazytoide dendritischer Zellen aus dem Knochenmark der Maus

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Die Beurteilung Retinal Microglial Phagozytische Funktion<em&gt; In Vivo</em&gt; Mit einem Durchfluss-Zytometrie basierten Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Ein Flow Cytometry-basierte Zytotoxizität Assay für die Beurteilung der menschlichen NK-Zell-Aktivität

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

Integration von Imaging Durchflusszytometrie und transkriptomischen Profilierung zu veränderten Endocytic CD1d Handel bewerten

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...