We are grateful to Prof. Zeng (IMCB, A*STAR) for supporting this work. This study was supported by the Youth Foundation of National Natural Science Foundation of China (NSFC) (82202231), and the Medical and Health Science and Technology Project of Zhejiang Province, China (2021KY110,2024KY824).

1. Detection of EGFR and VEGFR expression in target cells
NOTE: Use western blotting to detect the expression of the target antigen in the target cells.
<ol>
	<li>Sample preparation
	<ol>
		<li>Culture the cells (BHT-101 and SW-1736 human thyroid cancer cell lines) in T75 flasks using Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% antibiotics (Penicillin-Streptomycin).</li>
		<li>Harvest the cells when they are 80% confluent by removing the culture media and washing the cells with PBS. Add 5 mL of PBS and scrap the cells using a cell scraper to remove the cells from the culture flask. Transfer the cells into a 15 mL conical tube.</li>
		<li>Count the cells with a cell counter.</li>
		<li>Aliquot 5 &#215; 106 of BHT-101 and SW-1736 cells into 15 mL conical tubes.</li>
		<li>Spin down the cells at 135 &#215; g for 5 min at room temperature. Remove the supernatant PBS.</li>
		<li>Lyse the cell pellet by adding 100 &#181;L of RIPA lysis buffer containing protease-phosphatase inhibitors for 5 min on ice. Transfer the lysate to a 2 mL tube, centrifuge at 15,000 &#215; g for 15 min, and transfer the supernatant into new 2 mL tube.</li>
		<li>Measure the protein concentration of the cell lysate by using a bicinchoninic acid (BCA) assay kit.</li>
		<li>Aliquot 70 &#181;g of protein from cell lysate from each sample, add 2x Laemmli sample buffer supplemented with 10% 2-mercaptoethanol, and boil the samples at 100 &#176;C for 5 min for denaturation of samples.</li>
	</ol>
	</li>
	<li>Gel electrophoresis and membrane transfer
	<ol>
		<li>Prepare 8% sodium dodecyl sulfate (SDS) polyacrylamide gel using distilled water (4.7 mL), 30% acrylamide (2.7 mL), 1.5 M Tris Buffer, pH 8.8 (2.5 mL), 10% sodium dodecyl sulfate (0.1 mL), 10% ammonium persulfate (100 &#181;L), and TEMED (10 &#181;L).</li>
		<li>Run 70 &#181;g of the denatured samples in the 8% SDS-polyacrylamide gel at 80 V for 20 min and at 120 V for 100 min.</li>
		<li>Transfer the protein on the gel to a polyvinylidene fluoride (PVDF) membrane at 100 V for 90 min.</li>
		<li>After transferring, block the membrane with 7% skim milk diluted in TBS-T buffer (20 mM Tris pH 7.6, 140 mM NaCl, 0.2% Tween-20) for 1 h.</li>
	</ol>
	</li>
	<li>Antibody incubation
	<ol>
		<li>Incubate the blocked membrane with mouse anti-Epidermal Growth Factor Receptor (EGFR) and rabbit anti-Vascular Endothelial Growth Factor Receptor (VEGFR) monoclonal antibodies (diluted at 1:1,000 in 7% skim milk) overnight at 4 &#176;C.</li>
		<li>After incubation, wash the membrane thoroughly with Tris-buffered saline-Tween (TBS-T) buffer (20 mM Tris pH 7.6, 140 mM NaCl, 0.2% Tween-20) for 7 min and repeat 4x.</li>
		<li>Incubate the membrane with the HRP-conjugated secondary antibody (diluted at 1:4,000 in 7% skim milk (TBS-T)) for 1 h.</li>
		<li>After incubation, wash the membrane thoroughly with TBS-T buffer for 7 min and repeat 4 times.</li>
	</ol>
	</li>
	<li>Protein detection
	<ol>
		<li>Incubate the membrane in chemiluminescent HRP substrate prepared by mixing 1 mL of HRP substrate peroxide solution with 1 mL of HRP substrate Luminol Reagent (both reagents are from the chemiluminescent substrate kit) for 2 min.</li>
		<li>Visualize the membrane using an imaging system.</li>
	</ol>
	</li>
</ol>
2. Preparation of target cells
<ol>
	<li>Culture the target T cells, BHT-101 and SW-1736 cells exponentially until 80% confluent in RPMI media supplemented with 10% FBS, 1% L-glutamine, and 1% antibiotics (Penicillin-Streptomycin) in T-75 flasks.</li>
	<li>After removing the media, wash the cells once with PBS. Incubate the cells with 1 ml of non-enzymatic cell dissociation buffer for 5 min to dislodge the adherent cells. Add PBS to stop the reaction of the non-enzymatic cell dissociation buffer. Spin down the cells at 135 &#215; g for 5 min and add 5 mL of PBS. 
	NOTE: Here, we use a non-enzymatic cell dissociation buffer to preserve the integrity of membrane surface protein.</li>
	<li>Count the cells and seed them at 15,000 cells/wells in 96-well, white polystyrene microplates with a clear, flat bottom.</li>
</ol>
3. Preparation of varying concentrations of the therapeutic antibody
<ol>
	<li>To follow this protocol, use Cetuximab (chimeric anti-EGFR antibody) to bind EGFR and use Bevacizumab (humanized anti VEGF-A antibody) as a negative control.</li>
	<li>Prepare ADCC Bioassay buffer by adding 1.4 mL of low IgG serum into 33.6 mL of RPMI-1640 (supplied in the kit).</li>
	<li>Use ADCC Bioassay buffer for the dilution of antibodies. Prepare 400 &#181;L of each antibody in three different concentrations: 30 &#181;g/mL, 3 &#181;g/mL, and 0.3 &#181;g/mL (3x concentration) to get the final concentration of 10 &#181;g/mL, 1 &#181;g/mL, and 0.1 &#181;g/mL for wide-spectrum coverage to detect the optimal dose. 
	NOTE: Final antibody concentrations are based on 25 &#181;L of cultured target cells, 25 &#181;L of effector cells, and 25 &#181;L of antibody in each well of a 96-well plate.</li>
</ol>
4. Preparation of effector cells
<ol>
	<li>Store the effector cells at -80 &#176;C before use.</li>
	<li>Preheat the ADCC Bioassay Buffer in a 37 &#176;C water bath for at least 30 min before use.</li>
	<li>Thaw the ADCC Bioassay Effector cells from -80 &#176;C cold storage by placing them in a 37 &#176;C water bath (approximately 2-3 min). Gently rock and visually inspect the vial, but do not invert it during the thawing process.</li>
	<li>Transfer 630ul of Effector cells to a 15 mL tube containing 3.6 mL of ADCC Assay Buffer. Mix well by gently inverting the tube 2x.</li>
</ol>
5. Incubation of effector cells with antibody and target cells
<ol>
	<li>After overnight incubation, remove the media from the target cells (15,000 cancer cells per well) and add 25 &#181;L of the ADCC Bioassay buffer and 25 &#181;L of Cetuximab (EGFR antagonist) and Bevacizumab (VEGF antagonist) to get the final concentration of 0.1 &#181;g/mL, 1 &#181;g/mL, or 10 &#181;g/mL of the target cells in each well according to Figure 2.</li>
	<li>Add 75,000 effector cells in 25 &#181;L of ADCC bioassay buffer per well into the 96-well microplate containing target cells. The wells labeled as No mAb act as no antibody control (Figure 2).</li>
	<li>Add ADCC Bioassay buffer to the wells labeled as AB (ADCC Bioassay buffer) for blank control.</li>
	<li>Incubate the plate for 6 h.</li>
</ol>
6. Quantitative readout of ADCC activity
<ol>
	<li>Prepare the luciferase assay reagent 4 h before the measurement by adding Luciferase Assay buffer to the Luciferase Assay Substrate (lyophilized).</li>
	<li>After 6 h of incubation of target cells, antibody, and effector cells, add 75 &#181;L of the luciferase assay reagent to each well and incubate for 30 min.</li>
	<li>Following the incubation period, measure the luminescence signal in each well by using a luminometer, providing a quantitative readout of ADCC activity.</li>
	<li>For data analysis, calculate the fold Induction as follows: 
	Fold induction = RLU (antibody induced - background) / RLU (no antibody control - background). 
	Where RLU is relative luminescence units; Antibody induced are wells B3 to B8 and C3 to C8; No antibody control = B2 and C2; Background = Average RLU from well A2 to A5 (Figure 2).</li>
</ol>

Thyroid Cancer Cells as Effectors in an ADCC Bioassay for Cancer Immunotherapy

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	<li>Zahavi, D., AlDeghaither, D., O'Connell, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+antibody-dependent+cell-mediated+cytotoxicity:+a+strategy+for+improving+antibody-based+immunotherapy.">Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy.</a> Antib Ther. 1 (1), 7-12 (2018).</li><li>Fenis, A., Demaria, O., Gauthier, L., Vivier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+immune+cell+engagers+for+cancer+immunotherapy.">New immune cell engagers for cancer immunotherapy.</a> Nat Rev Immunol. 24 (7), 471-486 (2024).</li><li>Pinto, S., Pahl, J., Schottelius, A., Carter, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reimagining+antibody-dependent+cellular+cytotoxicity+in+cancer:+the+potential+of+natural+killer+cell+engagers.">Reimagining antibody-dependent cellular cytotoxicity in cancer: the potential of natural killer cell engagers.</a> Trends Immunol. 43 (11), 932-946 (2022).</li><li>Ochoa, M. 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=Antibody-dependent+cell+cytotoxicity:+immunotherapy+strategies+enhancing+effector+NK+cells.">Antibody-dependent cell cytotoxicity: immunotherapy strategies enhancing effector NK cells.</a> Immunol Cell Biol. 95 (4), 347-355 (2017).</li><li>Wang, W., Erbe, A. K., Hank, J. A., Morris, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cell-mediated+antibody-dependent+cellular+cytotoxicity+in+cancer+immunotherapy.">NK cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy.</a> Front Immunol. 6, 368 (2015).</li><li>Chung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+in+vitro+antibody-dependent+cell-mediated+cytotoxicity+activity+of+therapeutic+antibodies+-+impact+of+effector+cells.">Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.</a> J Immunol Methods. 407, 63-75 (2014).</li><li>Shimasaki, N., Jain, A., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cells+for+cancer+immunotherapy.">NK cells for cancer immunotherapy.</a> Nat Rev Drug Discov. 19 (3), 200-218 (2020).</li><li>Cheng, Z. 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=Development+of+a+robust+reporter-based+ADCC+assay+with+frozen,+thaw-and-use+cells+to+measure+Fc+effector+function+of+therapeutic+antibodies.">Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies.</a> J Immunol Methods. 414, 69-81 (2014).</li><li>Parekh, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+antibody-dependent+cell-mediated+cytotoxicity-reporter+gene+assay.">Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay.</a> MAbs. 4 (3), 310-318 (2012).</li><li>Hogarth, P. M., Pietersz, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+receptor-targeted+therapies+for+the+treatment+of+inflammation,+cancer+and+beyond.">Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond.</a> Nat Rev Drug Discov. 11 (4), 311-331 (2012).</li><li>Chung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+fucose+reducing+effects+in+a+humanized+antibody+on+Fcgamma+receptor+binding+and+antibody-dependent+cell-mediated+cytotoxicity+activities.">Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcgamma receptor binding and antibody-dependent cell-mediated cytotoxicity activities.</a> MAbs. 4 (3), 326-340 (2012).</li><li>. ADCC Reporter Bioassay Core Kit Technical Manual Available from: <a target="_blank" href="https://www.promega.sg/-/media/files/resources/protocols/technical-manuals/101/adcc-reporter-bioassay-core-kit-protocol.pdf?rev=bec36264c0b6470591ded081377d207d&amp;sc_lang=en">https://www.promega.sg/-/media/files/resources/protocols/technical-manuals/101/adcc-reporter-bioassay-core-kit-protocol.pdf?rev=bec36264c0b6470591ded081377d207d&amp;sc_lang=en</a> (2023)</li><li>Miller, A. S., Tejada, M. L., Gazzano-Santoro, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+measuring+antibody-dependent+cell-mediated+cytotoxicity+in+vitro.">Methods for measuring antibody-dependent cell-mediated cytotoxicity in vitro.</a> Methods Mol Biol. 1134, 59-65 (2014).</li><li>Lo Nigro, 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=NK-mediated+antibody-dependent+cell-mediated+cytotoxicity+in+solid+tumors:+biological+evidence+and+clinical+perspectives.">NK-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: biological evidence and clinical perspectives.</a> Ann Transl Med. 7 (5), 105 (2019).</li><li>G&#243;mez Rom&#225;n, V. R., Murray, J. C., Weiner, L. M., Ackerman, M. E., Nimmerjahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibody Fc. , 1-27 (2014).</li></ol>

All authors declare no conflicts of interest.

Here, we have presented the ADCC Bioassay method for assessing the ADCC reaction of a therapeutic antibody. The method is straightforward and employs a simple "add-mix-read" format for measurement. 
Before doing the experiment, the expression of the target antigen in the target cells must be confirmed by either flow cytometry or western blotting. Flow cytometry will be a better tool to detect the surface antigen. However, using flow cytometry can stress the cells, causing apoptosis and affecti...

Antibody-dependent, cell-mediated cytotoxicity (ADCC) is an important mechanism by which antibodies exert immune-mediated cell-killing effects1,2,3. The immune cells are activated by binding to the therapeutic antibody, which interacts with surface antigens of the target cells to release granzymes, perforin, leading to the target cell death. These immune cells include natural killer (NK) cells and neutrophils2,3,4,5,6,7. The ADCC assay has become an important tool to evaluate the efficacy of therapeutic antibody8,9.
In the conventional ADCC assay, peripheral blood mononuclear cells (PBMCs) or natural killer cells are used as effector cells to monitor the efficacy of a therapeutic antibody by quantitating the target&#39;s cell death rate. Our method uses an ADCC bioassay kit that includes genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. We then quantify the binding of the surface antigen on the target cells with the antibody and the effector cells. This method is based on the ADCC reaction occurring in a short period without requiring human PBMC cells. The experimental steps include the co-culture of effector cells with target cells in the presence of therapeutic antibodies.
During incubation, the therapeutic antibody binds to the target antigen on the surface of the target cells, which leads to the binding of the effector cells and the Fc fragment of an antibody. This activates the NFAT response element and releases luminescence signals for the quantitative assessment of the ADCC reaction.
Before performing the experiment, the expression of the target antigen in the target cells must be confirmed by either flow cytometry or western blotting. Target cells are cultured and passaged into 96-well plates for 24 h before the experiment. Different concentrations of a therapeutic antibody are added together with different cell counts of effector cells to achieve the calculated effector-to-target cell ratio.
Key steps in this method include (1) preparation of target cells and effector cells, (2) effector-to-target cell ratios, (3) Preparation of different concentrations of the antibody, and (4) varying duration of incubation. After the incubation, luminescence signals are measured using a luminometer, providing a quantitative readout of ADCC activity. Compared to other methods for measuring ADCC, this method is relatively simple to operate, and the results are accurate.
The ADCC reporter bioassay indicates the binding of the target antigen, therapeutic antibody, and immune cells in the ADCC pathway activation. This binding activates gene transcription through the NFAT pathway in the effector cells-engineered Jurkat cells with stably expressing Fc&#947;RIIIa receptor, the V158 (high-affinity) variant. The NFAT response element mediates the expression of luciferase in the effector cells10,11. The biological activity of the antibody in the Mechanism of action (MOA) of ADCC is quantified through the luciferase signal produced from the NFAT pathway. Luciferase signal in the effector cells-Fc&#947;RIIIa receptor-expressing Jurket cells-is quantified using a luminescence reader (Figure 1). The signal-to-noise ratio of the assay is high.

<table><tbody><tr><td>0.5% Trypsin-EDTA</td><td>Gibco</td><td>15400-054</td><td>Dilute 10x in PBS to make 0.05% Trypsin</td></tr><tr><td>1x Tris Buffer Saline (TBS)</td><td>1st BASE</td><td>BUF-3030-1X1L</td><td>For membrane washing in western blotting</td></tr><tr><td>1.5 M Tris Buffer, pH 8.8</td><td>1st BASE</td><td>BUF-1419-1L-pH8.8</td><td>For SDS gel preparation</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M7522-100ML</td><td>For sample preparation of western blotting</td></tr><tr><td>30% Acrylamide/Bis solution</td><td>Bio-Rad</td><td>#1610158</td><td>For SDS gel preparation</td></tr><tr><td>4x Laemmli Buffer</td><td>Bio-Rad</td><td>#1610747</td><td>For sample preparation of western blotting</td></tr><tr><td>96-well white polystyrene microplate with clear flat bottom</td><td>Corning Incorporated</td><td>3610</td><td>For ADCC assay</td></tr><tr><td>ADCC Bioassay Effector cells (0.65 mL)</td><td>Promega</td><td>G7011</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 1 vial</td></tr><tr><td>ADCC reporter bioassay core kit</td><td>Promega</td><td>G7010</td><td>Mentioned as ADCC bioassay kit for ADCC assay in this experiment</td></tr><tr><td>Ammonium Persulfate</td><td>Sigma Aldrich</td><td>A3678-25G</td><td>For SDS gel preparation</td></tr><tr><td>Bevacizumab (Humanized Anti VEGF-antibody)</td><td>MVASI</td><td>-</td><td>Use as negative control antibody in ADCC asssay</td></tr><tr><td>BHT-101</td><td>Leibniz Institute DSMZ</td><td>ACC279</td><td>Human anaplastic papillary thyroid cancer cell line&amp;nbsp;</td></tr><tr><td>Bio-Glo Luciferase Assay Buffer</td><td>Promega</td><td>G7941</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 10 mL</td></tr><tr><td>Bio-Glo Luciferase Assay Substrate (Lyophilized)</td><td>Promega</td><td>G7941</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 1 vial</td></tr><tr><td>Cell scraper</td><td>GenFollower</td><td>GD00235</td><td>To remove cell from culture flask</td></tr><tr><td>Cetuximab (Chimeric anti-EGFR antibody)</td><td>ERBITUX</td><td>-</td><td>Use as therapeutic antibody in ADCC assay</td></tr><tr><td>Chemiluminescent HRP substrate</td><td>Merck Millipore</td><td>WBKLS0500</td><td>For protein detection in western blotting</td></tr><tr><td>Distilled water</td><td>Gibco</td><td>15230-162</td><td>For SDS gel preparation</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td>Culture media supplement</td></tr><tr><td>iBright CL1500 imaging system</td><td>Thermo Scientific</td><td>2462621100038</td><td>For protein detection in western blotting</td></tr><tr><td>L-glutamine, 200 mM</td><td>Gibco</td><td>25030-081</td><td>Culture media supplement</td></tr><tr><td>Low IgG Serum</td><td>Promega</td><td>G7110</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 4 mL</td></tr><tr><td>Megafuge 8R</td><td>Thermo Scientific</td><td>42876589</td><td>Centrifuge</td></tr><tr><td>Mouse anti-EGFR monoclonal antibodies</td><td>BD Biosciences</td><td>610016</td><td>Primary antibody in western blotting</td></tr><tr><td>Mouse anti-VEGFR monoclonal antibodies</td><td>BD Biosciences</td><td>571194</td><td>Primary antibody in western blotting</td></tr><tr><td>non-enzymatic cell dissociation buffer</td><td>Sigma Aldrich</td><td>C5789-100ML</td><td>For cell harvesting from T75 flask</td></tr><tr><td>Penicillin-Streptomycin</td><td>PAN Biotech</td><td>P06-07100</td><td>Antibacterial for culture media</td></tr><tr><td>Phosphate Buffered Saline (PBS), pH 7.2, Sterile filtered</td><td>1st BASE</td><td>CUS-2048-1x1L</td><td>Use as washing solution for cells</td></tr><tr><td>Pierce BCA assay kit</td><td>Thermo Scientific</td><td>23225</td><td>To measure protein concentration</td></tr><tr><td>Protease and phosphatase inhibitor</td><td>Thermo Scientific</td><td>A32959</td><td>For protein digestion in sample preparation for western blotting</td></tr><tr><td>PVDF membrane (Immobilin-P)</td><td>Merck Millipore</td><td>IPVH00010</td><td>For protein transfer in western blotting</td></tr><tr><td>Rabbit anti-mouse IgG, Fc&amp;gamma; HRP-conjugated secondary antibody</td><td>Jackson ImmunoResearch</td><td>315-035-046</td><td>Secondary antibody in western blotting</td></tr><tr><td>Roswell Park Memorial Institute (RPMI) medium</td><td>Capricorn Scientific</td><td>RPMI-XA</td><td>Cell culture media</td></tr><tr><td>RPMI-1640</td><td>Promega</td><td>G7080</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 36 mL</td></tr><tr><td>Skim milk powder</td><td>Merck Millipore</td><td>70166-500G</td><td>For membrane blocking in western blotting</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>1st BASE</td><td>BIO-2050-500g</td><td>For SDS gel preparation</td></tr><tr><td>SW-1736</td><td>Cytion</td><td>300453</td><td>Human thyroid squamous cell cancer cell line</td></tr><tr><td>T75 culture flasks</td><td>SPL Lifesciences</td><td>70075</td><td>Cell culture flask</td></tr><tr><td>Tecan Multimode Reader model Spark 10M</td><td>Tecan</td><td>1607000294</td><td>for luminicence quantification</td></tr><tr><td>TEMED</td><td>Bio-Rad</td><td>#1610801</td><td>For SDS gel preparation</td></tr><tr><td>Tween-20</td><td>Promega</td><td>H5151</td><td>For membrane washing in western blotting</td></tr><tr><td>Vi-cell XR cell viability analyzer</td><td>Beckman Coulter</td><td>AL15072</td><td>Cell counter</td></tr></tbody></table>

assessing antibody-dependent, cell-mediated cytotoxicity in cancer cells using antibody-dependent cell-mediated cytotoxicity reporter bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

The expression of EGFR and VEGFR in the target BHT-101 and SW-1736 cells was detected using western blotting. EGFR expression was detected in both BHT-101 and SW-1736 cells but not VEGFR expression (Figure 3).
Using the ADCC bioassay kit, we detected the ADCC reaction of the anti-EGFR antibody, cetuximab, using EGFR-positive cell lines, BHT-101 and SW-1736, as target cells. Bevacizumab, a VEGF inactivator, was used as a negative control antibody. Different concent...

Author Spotlight: Optimizing Antibody-Based Cancer Treatments via Antibody-Dependent, Cell-Mediated Cytotoxicity Assay

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

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Immunology and Infection

281 Views.  A*STAR 61 Biopolis Drive. Het is algemeen bekend dat antilichaamafhankelijke, celgemedieerde cytotoxiciteit een belangrijk mechanisme is waarmee antilichamen immuungemedieerde bepaalde effecten uitoefenen. Daarom willen we een betere methode vinden om de werkzaamheid van een antilichaamgeneesmiddel bij de behandeling van kanker te testen, door ADCCSA te gebruiken. Het protocol is snel en duidelijk.Door gebruik te maken van een eenvoudig mix- en leesprotocol kunnen we binnen é?...

Video: Auteur Spotlight: Optimalisatie van op antilichamen gebaseerde kankerbehandelingen via antilichaamafhankelijke, celgemedieerde cytotoxiciteitstest

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

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

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

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

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 ...

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 ...

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

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

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 ...

Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or activating an endogenous regulator of the immune system have the potential to cause serious immune-related adverse events such as cytokine release syndrome (CRS). It is, therefore crucial to assess the safety profile of such drugs with a combination of in vivo and in vitro experiments before first-in-human dose administration. Cytokine release assays (CRAs), where the proposed antibody therapeutic is co-cultured with human immune cells (such as peripheral blood mononuclear cells (PBMCs), whole blood, or otherwise) and the amount of inflammatory cytokine produced is measured, are critical for hazard identification. However, different labs using different control antibodies can threaten the harmonization of CRAs, and clinically relevant controls (such as TGN1412) can be difficult to source, which can lead to less accurate or reliable results or data which are difficult to compare between laboratories. The inclusion of positive and negative controls in a CRA can ensure the accuracy and reliability of the results. The National Institute for Biological Standards and Control (NIBSC) has produced a panel of lyophilized antibody controls intended for use in various CRA platforms to harmonize results across various laboratories and assay methods. A set of three different positive control antibodies include anti-CD52, anti-CD3, and anti-CD28 superagonist (SA), which are known to induce dose-dependent CRS in patients. Each antibody is provided with an isotype-matched negative control antibody. This panel of reference reagents has previously been shown to have good inter-lab reproducibility and are suitable controls to increase the confidence and robustness of safety data from a variety of CRA platforms.

The use of cytokine release assay reference reagents allows for more reproducible and standardized in vitro safety profiles of immunotherapeutic monoclonal antibodies. Here we describe how cytokine release assays can be used alongside a reference reagent panel to predict the safety of some therapeutic monoclonal antibodies.

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or ...

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.

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

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