TITLE: In vitro  pmel-1 T cell-mediated cytotoxicity assay with CytoTox-ONE Homogenous Membrane Integrity Assay (Promega)
AUTHORS: Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Jeff Hammerbacher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>A plate-based assay to estimate the cytotoxic activity of pmel-1 T cells against target cancer cells. </span><a href="https://www.promega.com/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/cytotox_one-homogeneous-membrane-integrity-assay/?catNum=G7890" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">The CytoTox-ONE Homogeneous Membrane Integrity Assay from Promega</span></a><span> (Madison, WI) is a lactate dehydrogenase (LDH) release-based assay. Dying cells have damaged cell membranes causing cytosolic components, such as LDH, to leak into the culture media. The CytoTox-ONE assay uses LDH in the culture media to convert resazurin to fluorescent resofurin. The resulting fluorescent signal is proportional to the number of non-viable cells in a sample. One advantage to using this assay for T cell-mediated cytotoxicity is that the cancer cells do not need to be removed from the wells to measure cell death, which is especially convenient with adherent cancer cell types that would require trypsinization. However, we have found that the assay produces noisy data and a set of control wells with T cells alone is required to subtract background, overall increasing the number of replicates that are necessary.  We had consistent results when culturing the pmel-1 T cells with hgp100 peptide-pulsed MC38 colon cancer cells. But we had inconsistent results using pmel-1 T cells and their natural target, B16-F10 melanoma cells. This setup could be adapted for T cells other than pmel-1 and their target cells. The ratio of T cells to cancer cells and the duration of co-culture would have to be optimized.</span></div></div></div>

[STEPS]
?. The day before adding T cells to co-culture with the target cancer cells, plate the target cells in a 96 well plate at 25,000 cells per well in DMEM with 10 % FBS.An example plate layout is below:Plate the target cells in columns 2-7. Leave the rest of the wells empty.Each column has 6 replicates (we do not recommend reducing the number of replicates because the resulting data is too noisy)One plate per condition being tested is required (in this case, the minimum will be two plates, one will have the target cells pulsed with the peptide, one with unpulsed cells as a negative control).Sample plate layout:       Incubate the target cells overnight to allow them to adhere to the plate.
?. Prepare the target cells for co-culture:If required, pulse the target cells in one plate with 1 uM hgp100 (peptide for   at Wash the peptide out 3x with T cell mediaPrepare T cells for co-culture (the day post-activation will depend on your experiments).Centrifuge the T cells at   for    and resuspend in T cell media supplemented with IL-2 (200IU/ml) to obtain a concentration of 2 million cells per mlCo-culture T cells with target cells:Aspirate culture media from 96 well platePrepare T cells for co-culture (the day post-activation will depend on your experiments).Centrifuge the T cells at  for   and resuspend in T cell media supplemented with IL-2 (200IU/ml) to obtain a concentration of 2 million cells per mlCo-culture T cells with target cells:Pipette   T cell media into columns 3-7 and 9-12 (leaving columns 2 and 8 empty)Pipette  of T cell suspension into columns 2, 3, 8 and 9Serial dilute the T cells in the plate: there will now be 200 ul total volume in columns 3 and 9. Starting from column 3, pipette up and down  3 times to mix. Then transfer  to column 4. Pipette up and down to mix in column 4 and transfer  to column 5. Pipette up and down to mix in column 5 and remove  and discard. Repeat starting at column 9 to make the T cell dilutions without cancer cells. Incubate plates for  at   .
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Centrifuge: 350 34
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?. Perform CytoTox-ONE Homogenous Membrane Integrity Assay:Thaw assay buffer and stop solution from CytoTox-ONE Homogenous Membrane Integrity Assay  kit and bring to room temperature. Thaw lysis solution and make a 1:5 dilution of lysis solution in PBS - total volume 100 ul per plate; this is good for 1 plate (80 ul PBS + 20 ul lysis solution)Take plate/s out of incubator and place in hood As soon as taking plates out of the incubator add    diluted lysis solution to each well in column 7 to lyse the cells for the maximum LDH release control. Make sure to mix the lysis buffer into the well by pipetting up down.Allow plates to sit in hood for   to equilibrate to room temperatureAdd 11 ml Assay Buffer into one substrate bottle and mixPour the assay buffer/substrate mixture into a reagent reservoir and using multichannel pipette, pipette   into each well to assay in the plate, shake plate gently to mixCover from light in hood and incubate for   Add   stop solution to each well in the same order as the assay buffer was added, shake plate gently to mixDirectly read plates on a fluorescence plate reader (excitation 560, emission 590) in the order that they were prepared inThaw lysis solution and make a 1:5 dilution of lysis solution in PBS - total volume 100 ul per plate; this is good for 1 plate (80 ul PBS + 20 ul lysis solution)Take plate/s out of incubator and place in hood As soon as taking plates out of the incubator add    diluted lysis solution to each well in column 7 to lyse the cells for the maximum LDH release control. Make sure to mix the lysis buffer into the well by pipetting up down.Allow plates to sit in hood for   to equilibrate to room temperatureAdd 11 ml Assay Buffer into one substrate bottle and mixPour the assay buffer/substrate mixture into a reagent reservoir and using multichannel pipette, pipette   into each well to assay in the plate, shake plate gently to mixCover from light in hood and incubate for   Add   stop solution to each well in the same order as the assay buffer was added, shake plate gently to mixDirectly read plates on a fluorescence plate reader (excitation 560, emission 590) in the order that they were prepared inAs soon as taking plates out of the incubator add   diluted lysis solution to each well in column 7 to lyse the cells for the maximum LDH release control. Make sure to mix the lysis buffer into the well by pipetting up down.Allow plates to sit in hood for  to equilibrate to room temperatureAdd 11 ml Assay Buffer into one substrate bottle and mixPour the assay buffer/substrate mixture into a reagent reservoir and using multichannel pipette, pipette   into each well to assay in the plate, shake plate gently to mixCover from light in hood and incubate for   Add   stop solution to each well in the same order as the assay buffer was added, shake plate gently to mixDirectly read plates on a fluorescence plate reader (excitation 560, emission 590) in the order that they were prepared inPour the assay buffer/substrate mixture into a reagent reservoir and using multichannel pipette, pipette  into each well to assay in the plate, shake plate gently to mixCover from light in hood and incubate for Add  stop solution to each well in the same order as the assay buffer was added, shake plate gently to mix
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?. Calculate percent cytotoxicity:((experimental-CMB)-(Tcellbackground-CMB)/(maxLDHrelease-CMB))X 100where experimental is the co-culture well (columns 2-6), CMB (culture medium background) is culture media with no cells (column 12), T cell background is T cells only at the different concentrations corresponding to the co-culture ratios (columns 8-11), and max LDH release is MC38 cells lysed with a detergent to release all of the LDH in the cells into the culture media (column 7).