Name: cell-based flow cytometry assay to measure cytotoxic activity
Uploaded: 2013-12-17T13:45:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity at JoVE.com

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

17.9K Views.  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.

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

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 ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Cancer Research

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.

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 ...

Medicine

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.

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

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, ...

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 ...

Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
One of the main functions of the cells of the immune system is to remove target cells that have been infected with viruses or have undergone transformation into a tumor cell. In vitro assays for measuring the cytotoxic capacity of immune cells have been a staple in laboratories for many years. These assays have been used to determine the ability of T cells, NK cells, or any other immune cell to kill target cells in an antigen-specific or -nonspecific manner. Death ligands (e.g., Fas ligand or TRAIL), cytokines (e.g., IFNg or TNF), or cytotoxic granules (i.e., perforin/granzyme B) expressed by effector cells are some ways in which target cell death can be induced. With the explosion in tumor immunotherapy research in recent years, there is growing interest in finding agents to increase the cytotoxic activity of immune cells to improve patient outcomes. Conversely, some diseases are hallmarked by the overexuberant activity of immune cell cytotoxic activity, resulting in efforts to identify agents to temper these responses. Thus, having an assay in which the user can easily integrate any number of different effector cells, target cells, and/or response modifiers into the experimental design can serve as a valuable means of quickly assessing the cytotoxic capacity of effector cells and/or the responsiveness of the target cell.
These in vitro assays involve the mixing of different cell populations, as well as using a relatively low number of both effector and target cells. Thus, one necessity of the assay is to label the target cells in a manner that can easily be detected and quantitated, allowing the user to then determine the &#39;percent specific lysis&#39; mediated by the effector cells. Radioactivity - especially, chromium 51 (51Cr) in the form of Na251CrO4- is an inexpensive way to rapidly and nonspecifically label cellular proteins within the target cells (1). The short labeling and total assay times reduces the potential for significant changes in the number and/or phenotype of the target cells, which could influence the outcome of the assay. Upon the loss of membrane integrity of the target cells as a result of the cytotoxic activity of the effector cells, the 51Cr-labelled cellular proteins within the target cells are released into the culture supernatant, becoming available for quantitation. As with any assay examining the function of immune cells in vitro, there are a number of important considerations to consider improving the performance of the experiment. One of the most critical features is to use healthy effector (for maximal cytotoxic activity) and target (for maximal responsiveness and minimal spontaneous death/51Cr release) cells. Effector and target cell contact is required (leading to the common use of round-bottom 96-well plates to encourage cell-cell contact) (2). Finally, data analysis is dependent on the inclusion of positive and negative control target cell populations.
The following protocol will outline the steps for performing a standard 51Cr release assay to measure the cytotoxic ability of a population of effector cells, though a nonradioactive version using Europium has recently been developed. 51Cr is a powerful &#947;-radiation emitter. Consequently, the use of this assay requires proper radiation safety training, dedicated laboratory space, a gamma counter, and disposal of radioactive samples.
The general sequence of events in this assay are: 1) prepare 51Cr-labeled targets; 2) prepare effector cells and add to plate while target cells are labeling; 3) add labeled targets to plate; 4) incubate plate; 5) harvest supernatants; and 6) analyze data after running samples on counter. Samples are commonly prepared in triplicate, and then averaged to account for any subtle pipetting differences.
Proper PPE is important for this assay. Specifically, the user should wear a lab coat and gloves. Safety glasses may be required based on the laboratory or institution. There should be ample lead shielding for safe storage and use of the 51Cr during all steps. Finally, there should be dedicated lab space and equipment set aside for using 51Cr, including all the proper signage to indicate where samples with 51Cr are being kept and a Geiger counter equipped with gamma probe to survey the space for possible contamination.
In this lab exercise, we will determine the ability human peripheral blood mononuclear cells (PBMCs), (CpG stimulated vs. unstimulated) to kill melanoma cells, using human melanoma cell line WM793 as model and the chromium release assay.

Chromium Release Assay for Testing Cytotoxicity

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

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Immunology

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Source: McBride, J. E., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54779">Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.</a> J. Vis. Exp. (2017).
This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.

Test cytotoksyczności za pośrednictwem komórek NK Przygotowanie próbki do analizy cytometrycznej przepływowej

1.&#160;Preparation of Controls

	Transfer the following into separate and appropriately labeled 1.5 ml tubes:
	
		Add 500 &#181;L of fresh IMDM ...

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Immunodiagnostics

Cellular Immunodiagnostics

An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells

Source: Hsiao, C. H., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60420">Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment.</a> J. Vis. Exp. (2020).
This video demonstrates an in vitro technique to measure the cytotoxic ability of cytokine-induced killer (CIK) cells against cancer cells. Upon co-culturing target cancer cells with CIK cells, the dead cancer cells are identified using a fluorescent viability dye and are quantified using flow cytometry, estimating the cytotoxic effect of CIK cells.

Test in vitro do badania zdolności cytotoksycznych komórek zabójczych indukowanych cytokinami

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...