We thank National Cancer Institute IMAT program from NIH for funding this work [R33 CA174616-01A1].

1. Target Cell Preparation
<ol>
	<li>Maintain target cells (e.g. Raji lymphoma cells) in growth media (RPMI 1640 culture medium, with 10% heat-inactivated fetal bovine serum (FBS), 4nM L-glutamine, and 500 IU/ml penicillin/ streptomycin) at 37oC in a 5% CO2 incubator.</li>
	<li>Centrifuge the sample to pellet the target cells in a 15 ml tube. Centrifugation time varies with different cell types; centrifuge for 3 min at 468 x g for Raji cells.</li>
	<li>First aspirate any bubbles formed on surface of the supernatant, and remove the entire supernatant.</li>
	<li>Add 10 ml of PBS to the tube. Re-suspend the cells by pipetting the solution up and down several times to break up the cell clump.</li>
	<li>Centrifuge the sample for 3 min at 468 x g.</li>
	<li>Aspirate any bubbles formed on the surface of the supernatant, and remove the entire supernatant.</li>
</ol>
2. Preparation of Cell Microarrays
<ol>
	<li>Obtain cell attachment 96 well plate kits or single cell array 8 well chamber slides (see table of materials/reagents).</li>
	<li>Apply 1.2 &#956;l&#160;of the Activator to the DNA reagent from the kit. Cap the vial, invert it, and gently shake to mix the solution.</li>
	<li>Allow the solution to react for 20 min at RT.</li>
	<li>Apply the mixed solution to the target cells pellet (in the 15 ml tube).</li>
	<li>Add green fluorescence cytosolic dyes at a final concentration of 500 nM.</li>
	<li>Incubate the target cells with the mixed solution and the dye for 30 min at RT on an orbital shaker.</li>
	<li>Wash the cells twice with 5 ml PBS, and resuspend the cells in 10 ml growth media.</li>
	<li>Apply 100 &#181;l of the solution from step 2.7 to each well. Count the cell number by using a hemacytometer. Make sure cell numbers range between 5&#215;104 to 1&#215;106 cells per well.</li>
	<li>After 10-15 min of incubation at RT, gently wash the samples twice with 200-300 &#181;l growth media to remove any unbound cells. 
	NOTE: About 5-10 &#181;l growth media remains in each well after washing.</li>
</ol>
3. Whole Blood Cytotoxicity Assays
<ol>
	<li>Add 180 &#956;l of human whole blood to each sample well.</li>
	<li>Obtain Anti-CD20 antibody solution at 5 mg/ml, and perform serial dilution to make 50, 20, 10, 5, 2, 1, 0.5 &#956;g/ml of Anti-CD20 antibody solution in 1.5 ml tubes. Add 20 &#181;l of each concentration of Anti-CD20 antibody to each sample well making triplicates.</li>
	<li>Add Propidium iodide at a final concentration of 500 nM to each well.</li>
	<li>Incubate the resulting plate at 37&#176;C with 5% CO2 for 16 hr.</li>
	<li>Image the cells on the slides or 96 well plates with an automatic imaging system with 8X magnification.</li>
	<li>Use the cell counting program of the automatic imaging system to quantify the results. 
	NOTE: The software of the automatic imaging system provides simple step-by-step operations for cell counting. The protocol of using this software can be found at provider&#8217;s website.</li>
	<li>Select [Review Results] from the main page of the software.</li>
	<li>Select [The File Saved for The Samples].</li>
	<li>First gate FITC mean intensity &#62;50, then gate TxRed mean intensity &#62;20.</li>
	<li>Select [Plate Overview].</li>
	<li>Select [Export Tables]. 
	NOTE: The percentage cell killing formula of the program is shown below: 
	% target cell killing = (# of cells stained both green and red/ # of cells stained green)*100%</li>
</ol>

<ol>
	<li>Hutchinson, L., Kirk, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+drug+attrition+rates--where+are+we+going+wrong.">High drug attrition rates--where are we going wrong.</a> Nat Rev Clin Oncol. 8, 189-1890 (2011).</li><li>Moreno, L., Pearson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attrition+rates+be+reduced+in+cancer+drug+discovery.">Attrition rates be reduced in cancer drug discovery.</a> Informa healthcare. 8, 363-368 (2013).</li><li>Seok, J., 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=Inflammation+and+Host+Response+to+Injury,+Large+Scale+Collaborative+Research+Program.+Genomic+responses+in+mouse+models+poorly+mimic+human+inflammatory+diseases.">Inflammation and Host Response to Injury, Large Scale Collaborative Research Program. Genomic responses in mouse models poorly mimic human inflammatory diseases.</a> Proc Natl Acad Sci U S A. 110, 3507-3512 (2013).</li><li>Suntharalingam, G., 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=Cytokine+storm+in+a+phase+1+trial+of+the+anti-CD28+monoclonal+antibody+TGN1412.">Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412.</a> N Engl J Med. 355, 1018-1028 (2006).</li><li>Eastwood, 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=Monoclonal+antibody+TGN1412+trial+failure+explained+by+species+differences+in+CD28+expression+on+CD4++effector+memory+T-cells.">Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells.</a> Br J Pharmacol. 161, 512-526 (2010).</li><li>Hsiao, S. C., Liu, H., Holstlaw, T. A., Liu, C., Francis, C. Y., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+assays+for+quantifying+cytotoxicity+with+single+cell+resolution.">Real time assays for quantifying cytotoxicity with single cell resolution.</a> PLoS One. 8, 10-1371 (2013).</li><li>Wang, S. Y., Weiner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rituximab:+a+review+of+its+use+in+non-Hodgkin&#8217;s+lymphoma+and+chronic+lymphocytic+leukemia.">Rituximab: a review of its use in non-Hodgkin&#8217;s lymphoma and chronic lymphocytic leukemia.</a> Expert Opin Biol Ther. 8, (2008).</li><li>Dalle, S., Thieblemont, C., Thomas, L., Dumontet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+in+clinical+oncology.">Monoclonal antibodies in clinical oncology.</a> Anticancer Agents Med Chem. 8, 523-532 (2008).</li><li>Brunner, K. T., Mauel, J., Cerottini, J. C., Chapuis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assay+of+the+lytic+action+of+immune+lymphoid+cells+on+51-Cr-labelled+allogeneic+target+cells+in+vitro;+inhibition+by+isoantibody+and+by+drugs.">Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs.</a> Immunology. 14, 181-196 (1968).</li><li>Korzeniewski, C., Callewaert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-release+assay+for+natural+cytotoxicity.">An enzyme-release assay for natural cytotoxicity.</a> J Immunol Methods. 64, (1983).</li><li>Gerlier, D., Thomasset, N. J. <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+MTT+colorimetric+assay+to+measure+cell+activation.">Use of MTT colorimetric assay to measure cell activation.</a> Immunol Methods. 94, 57-63 (1986).</li><li>Toriello, N. M., 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=Integrated+microfluidic+bioprocessor+for+single-cell+gene+expression+analysis.">Integrated microfluidic bioprocessor for single-cell gene expression analysis.</a> Proc Natl Acad Sci U S A. 105, (2008).</li><li>Douglas, E. S., Hsiao, S. C., Onoe, H., Bertozzi, C. R., Francis, M. B., Mathies, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-barcode+directed+capture+and+electrochemical+metabolic+analysis+of+single+mammalian+cells+on+a+microelectrode+array.">DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.</a> Lab on a Chip. 9, 2010-2015 (2008).</li><li>Mayr, L. M., Bojanic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+trends+in+high-throughput+screening.">Novel trends in high-throughput screening.</a> Current opinion in pharmacology. 9, 580 (2009).</li><li>Zanella, F., Lorens, J. B., Link, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening:+seeing+is+believing.">High content screening: seeing is believing.</a> Trends in Biotechnology. 28, 237-245 (2010).</li><li>Coelho, J., P, L., Quinn, S., Murphy, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image+analysis+for+high-content+screening+and+analysis.">Automated image analysis for high-content screening and analysis.</a> J Biomol Screen. 15, 726-734 (2010).</li><li>Sundberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+and+ultra-high-throughput+screening:+solution-+and+cell-based+approaches.">High-throughput and ultra-high-throughput screening: solution- and cell-based approaches.</a> Current Opinion in Biotechnology. 11, 47 (2000).</li><li>Simmons, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+medicine+is+more+than+genomic+medicine:+confusion+over+terminology+impedes+progress+towards+personalized+healthcare.">Personalized medicine is more than genomic medicine: confusion over terminology impedes progress towards personalized healthcare.</a> PERS MED. 9, 85 (2012).</li></ol>

Authors have no competing financial interests.

WCA is a critical in vitro anti-cancer screening tool with single cell resolution12-16, ideally utilized after traditional target screenings such as CDC and ADCC assays9-11, and before preclinical animal tests. Currently, primary target screening assays such as CDC or ADCC assays are all performed in a simplified media or a buffer system. However, drug candidates that show efficacy in these simplified buffer system are not always effective in the more complex whole blood system. Therefore, WCA can bridge the gap between traditional target screenings and costly animal studies, reduce false positives, and thus prevent the preventable failures in animal tests or in human clinical trials. The WCA will be beneficial for the researchers working on the preclinical studies in order to test the drug efficacy in the content of human whole blood. Counting the dead and live cells using flow cytometry requires the complete lysis of red blood cells in order to detect target cells. The advantage of WCA technique over flow cytometry is that it can identify target cells without lysis of red blood cells. It is difficult to completely lyse all the red blood cells in the blood sample, and target cells are also partially lysed during the lysis procedure.
Even more exciting opportunities arise if screening panels can be generated using the live primary cells obtained from individual patients, paving the way to personalized cancer treatments, the evaluation of cell heterogeneity within a given tumor, and the identification of cells that are resistant to a given drug treatment. Moving the healthcare system to an approach that is &#8220;personalized, predictive, preventive and centered on the needs of the patient&#8221; is the future of medicine17,18. Numerous initiatives within the US Department of Health and Human Services, including the FDA, the Centers for Disease Control, the NIH, the Centers for Medicare and Medicaid Services, and the Health Resources and Services Administration exist to support initiatives to promote personalized care. The realization of personalized medicine depends on reliable technologies and products that can capture and hold any human cells while maintaining them in a relevant biological state. We can foresee applying WCA to screen drugs against cancer patient&#8217;s tumor cells in the matrix of his/her own blood for personalized medicine applications.
The critical steps in the protocol are the preparations of the cell array. The users need to remove all of the supernatant without losing the cells during the cell washing step. In addition, users need to do a short centrifugation if the liquid cannot be seen in the vials. Once DNA reagent solution has been prepared, it must be used with the cells within 30 min.
The cell array formation efficiency is cell type dependent. There have been more than 100 types of cells tested with this protocol; however, it is still possible that some specific cell types will not form the cell array efficiently. If no cell array forms, a higher concentration of DNA reagent is recommended to perform the same protocol to get better cell array formation.

Recent advances in the pharmaceutical industry have led to an increased interest in realizing the specific identifications of tumor cell antibodies and personalized cancer treatments; however, several obstacles are encountered in the process. Only 5% of agents that have anticancer activity in preclinical development are licensed after showing sufficient efficacy in phase II-III testing1,2. The preclinical strategies (both in vitro and in vivo) are suboptimal as many examples have shown that antitumor drugs behave differently in human and in laboratory animals mainly due to their different blood components3-5.
To address the necessity of an antitumor drug screening platform and to provide a check-point before costly animal experiments and clinical trials, a human whole blood cytotoxicity assay (WCA) is proposed for evaluating antitumor drug efficacy in a more relevant biological environment. The whole blood cytotoxicity assay can be used to evaluate the response of individual cells to antibodies and other drug candidates in human whole blood.
The WCA is developed by incorporating high-throughput cell positioning technology with high-throughput and high-content imaging6. By utilizing an automated imaging system, the number of both living and dead cells can be determined with a high degree of precision. Due to the fact that target cells are immobilized on the same focal plane, WCA is able to provide quantitative cytotoxicity analysis in real time without the removal of red blood cells. Moreover, the automatic imaging system provides several advantages such as that only target cells that have reached the specified criteria (e.g., fluorescently labeled cells, and cell morphology) are gated and processed. Also, it allows the production of 144 plates per day. Consequently, this imaging capability and throughput allows running of high-content and high-throughput experiments simultaneously. By combining WCA and the automated imaging system, high throughput quantitative cell cytotoxicity analysis can be achieved within a more biological relevant environment.

<table><tbody><tr><td>Cell attachment 96 well plate kits&amp;nbsp;</td><td>Adheren</td><td>AP9601</td><td></td></tr><tr><td>Suspension Single cell array 8 well chamber slide</td><td>Adheren</td><td>SS0801</td><td></td></tr><tr><td>Adherent Single cell array 8 well chamber slide</td><td>Adheren</td><td>SS0802</td><td></td></tr><tr><td>Lymphoma cell line CD20+</td><td>ATCC</td><td>CCL-86</td><td>Raji cells</td></tr><tr><td>Lymphoma cell line CD20-</td><td>ATCC</td><td>CRL-8083</td><td>MC/CAR</td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Life Technologies</td><td>11875-119</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo</td><td>SH30070.01HI</td><td></td></tr><tr><td>Peni/Strep</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Cytosolic dye</td><td>Life Technologies</td><td>C7025</td><td>Cell Tracker Green</td></tr><tr><td>Rituxan (Biosimilar)&amp;nbsp;</td><td>Eureka Therapeutics</td><td></td><td></td></tr><tr><td>Human whole blood</td><td>Allcells</td><td>WB001</td><td></td></tr><tr><td>Propidium Iodide</td><td>Sigma</td><td>P4170-10MG</td><td></td></tr><tr><td>Automatic imaging system</td><td>Molecular Devices</td><td>Contact Vendor</td><td>Cell Reporter</td></tr><tr><td>Cell counting program</td><td>Molecular Devices</td><td>Contact Vendor</td><td>Cell Reporter</td></tr></tbody></table>

real-time cytotoxicity assays in human whole blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

Anti-CD20 antibodies and lymphoma cells (Raji cells and MC/CAR) were chosen as a model system to demonstrate the whole blood cytotoxicity assay (WCA)7,8. Raji cells had high copy number of CD20 on the cell surface, while MC/CAR cells had low copy number of CD20 on their membrane. Targeted cells were first stained green with green fluorescence cytosolic dyes and arrayed on the 96-well plate. 10,000 - 50,000 target lymphoma cells were immobilized in each well. 180 &#181;l of freshly drawn human whole blood was added into each well. Anti-CD20 antibody was then added into each well of a 96 well plate with gradient concentrations. After 16 h of incubation at 37&#176;C, cell numbers were assessed quantitatively through the automatic imager by analyzing the number of green fluorescent cells and red fluorescent cells. Since the target lymphoma cells were all arrayed on the same focal plane, the fluorescence of each cell was detected directly even without removing the blood cells in the well.
As shown in Figure 1, the detection of the targeted Raji lymphoma cells (both alive and dead) was performed. Green dots in Figure 1 indicated the number of target cells presented. Dead cells (red dots) in Figure 1 were analyzed quantitatively through the intensity of the red fluorescence because they were stained fluorescent red with PI. Antibody cytotoxicity was analyzed through the ratio of dead cells (stained both green and red) to the cells presented (green dots). The number of fluorescent red dots decreased as the concentration of Anti-CD20 antibody decreased in concentration.
Each antibody concentration was tested independently in triplicates. For each antibody concentration, more than 400 target cells per well were imaged to provide the mean value and the standard deviation of cytotoxicity results. Based on our data, with 400 data points per antibody concentration, we obtained statistical results with p value &#60;0.01. The dose-dependent drug efficiency was obtained in Figure 2. The MC/CAR cells with low copy number of CD20 antigen were used as a control cell line. An EC50 value of 0.12 &#956;g/ml was obtained for the WCA result against Raji lymphoma cells, which was in the range with the value obtained in the regular antibody cytotoxicity assays including complement dependent cytotoxicity assay (CDC) or antibody-dependent cellular cytotoxicity (ADCC) assay.9-11
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51941/51941fig1highres.jpg" width="500" /> 
Figure 1. Antibody-based cytotoxicity measured in whole blood. Raji cells were labeled with a green cytosolic dye and then immobilized. PI was also added to access cell death. Dead cells were stained red by PI. Varying concentrations of Anti-CD20 antibody were added with the addition of whole blood. (a) 2.5 &#181;g/ml Anti-CD20 antibody 16 hr after, (b) 1 &#181;g/ml Anti-CD20 antibody 16 hr after, (c) 0.5 &#181;g/ml Anti-CD20 antibody 16 hr after, (d) 0.25 &#181;g/ml Anti-CD20 antibody 16 hr after , (e) 0.1 &#181;g/ml Anti-CD20 antibody 16 hr after, (f) 0.05 &#181;g/ml Anti-CD20 antibody 16 hr after, (g) 2.5 &#181;g/ml control antibody 16 hr after.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51941/51941fig2highres.jpg" width="500" /> 
Figure 2. Dose-dependent cytotoxicity against different cell lines in human whole blood. Raji was a CD20 positive lymphoma cells, and MC/CAR was a CD20 negative cell lines. The level of whole blood cytotoxicity was shown for different concentration of &#945;-CD20 antibody (Anti-CD20 antibody).

Watch this Scientific Journal Video about Real-time Cytotoxicity Assays in Human Whole Blood at JoVE.com

Real-time Cytotoxicity Assays in Human Whole Blood

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

real-time-cytotoxicity-assays-in-human-whole-blood

Nanyang Technological University

Research

JoVE Journal

Medicine

12.1K Views.  Adheren, Inc.  De volbloed cytotoxiciteit assay (WCA) is een cytotoxiciteit assay ontwikkeld door het opnemen van high-throughput cell technologie voor plaatsbepaling met fluorescentie microscopie en geautomatiseerde beeldverwerking. Hier beschrijven we hoe lymfoomcellen behandeld met een anti-CD20 antilichaam kan worden geanalyseerd real-time in humaan volbloed kwantitatieve cellulaire cytotoxiciteit analyse. 

Video: Real-time Cytotoxicity Assays in Human Whole Blood

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.

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 ...

Immunology and Infection

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Cancer Research

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.

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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.

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

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Optimized Whole Blood Immunoassays for Analyzing Fungal and Viral Immunity

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Author Spotlight: Advancing Immune Monitoring in Critical Care Patients Using Whole Blood Assays

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in ...

A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells

Source: Xi, B. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59033">A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a method for monitoring CAR T cell activity against cancer cells through real-time electrical impedance measurements. Tracking changes in impedance assesses the effectiveness of CAR T cells in inducing cancer cell cytolysis, providing insights into their cytotoxic capabilities.

1. Generation of CAR-encoding lentivirus
NOTE: Once the specific chimeric antigen receptor (CAR) T-cell plasmid construction is completed (CD47 and ...

Real-time Cytotoxicity Assays in Human Whole Blood

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