Feb 28, 2024

Public workspaceCytotoxicity Assay Protocol

DOI
dx.doi.org/10.17504/protocols.io.14egn35mpl5d/v1
  • Roshni Jaffery1,2,
  • Ningbo Zheng1,2,
  • Jiakai Hou1,2,
  • Ashley Guerrero1,2,
  • Si Chen1,2,
  • Chunyu Xu1,2,
  • Nicholas A. Egan1,2,
  • Ritu Bohat1,2,
  • Weiyi Peng1,2
  • 1Department of Biology and Biochemistry, University of Houston, Houston, TX, USA;
  • 2Aligning Science Across Parkinson's Collaborative Research Network
Sarfraz Ahmed
Open access
DOI: dx.doi.org/10.17504/protocols.io.14egn35mpl5d/v1
Protocol Citation: Roshni Jaffery, Ningbo Zheng, Jiakai Hou, Ashley Guerrero, Si Chen, Chunyu Xu, Nicholas A. Egan, Ritu Bohat, Weiyi Peng 2024. Cytotoxicity Assay Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn35mpl5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 28, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95903
Keywords: cytotoxicity assay, cancer cell lines, flow cytometry
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000312
Abstract
This is the protocol for cytotoxicity assay in cancer cell lines using flow cytometry.
Materials
1. Tumor cell line
2. Tumor-reactive T cells
3. Tumor cell medium: RPMI 1640 (Gibco 11875) + 10% Fetal Bovine Serum (FBS) complete mixture
4. T cell medium: 50ml complete MEM media (Gibco 11095080) + 500 IU/ml IL2 + add 50 µL of 1000X beta-mercaptoethanol (55mM) 5. 1X Phosphate-buffered saline (PBS)
6. FACS Buffer: 2% FBS in PBS
7. APC-conjugated rabbit anti-active caspase 3 Ab
8. BD Cytofix/Cytoperm‱ Fixation/Permeabilization Kit
9. Fixation/permeabilization solution
10.BD Perm/Wash‱ Buffer
Begin T cell culturing a week prior. Grow and culture tumor cell lines. Ensure the cells are not over-confluent. 
Split the tumor cells the day before the assay. Perform the cytotoxic assay as described in next steps.
Count tumor cells. Collect 3-5M cells for each cell line.
Centrifuge the cells and wash the pellet once with PBS. Ensure that all media is washed off as this can inhibit the staining.
Aspirate the supernatant and leave as little PBS as possible
Re-suspend cells in culture medium at the appropriate ratio for the experiment (1X106 / ml for 50,000 cells/50ul/well or about ~80% confluent) using 96 well round well plate with 50,000 cell tumor and in triplication.
Add 50 ul of T cell suspension diluted to achieve the desired E:T ratio. Below is an example of how to set up E:T ratios:
ABC
ET Ratio(s) T cells/50ul /well Tumor cells/50ul/well
0:1 0 (only with T cell medium) 5 x104 cells
1:1 5 x104 cells 5 x104 cells
3:1 1.5x105 cells 5 x104 cells

Centrifuge the mixed cells in the tubes at 300 rpm for 5 minutes to gently pellet the cells. Incubate for 3 hours at 370C.
Add 150ul PBS into the plate and then centrifuge at 2000rpm for 5min.
Remove the supernatant and add then 100ul/well Trypsin. The plate is incubated at 37C incubator for 3-5mins.
Add 100ul FACS buffer to stop the trypsinization and centrifuge at 2000rpm for 5 min.
Remove the supernatant carefully.
Fix and permeabilize cells using Cytofix/CytopermTM Kit (BD BioSciences) for 20 minutes at room temperature (100ul/well – mix well).
Wash with Perm/WashTM Buffer (150ul/well) at 2000 rpm for 5 min.
Prepare caspase antibody dilution such that each well gets stained with 2.5ul of APC-conjugated rabbit anti-active caspase 3 Ab in 50 ul Perm/WashTM Buffer. Mix gently and incubate for 30 min in the dark on ice or in the 4°C refrigerator.
Wash cells 1Xwith Perm/WashTM Buffer (150ul).
Resuspend the cell pallets into 100ul FACS buffer (2% FBS in 1X PBS).
Analyze the samples using flow cytometry.