I am currently performing co-culture experiments with murine CD8 T cells isolated from mouse spleens and with skin cancer cells (PDV) in a 24-well plate. I first isolate the CD8 T cells from spleen which are around 70% alive at time of isolation, mix them together with CD3/CD28 T Cell activation beads to activate the CD8 T cells, and plate them 2x10^5 T cells on top of 2x10^5 skin cancer cells that were plated the night before. The cancer cells are treated with mitomycin C prior to plating the T cells in order to halt cancer cell proliferation. Then I let them incubate in a tissue culture incubator with standard parameters (37 degrees Celsius and 5% CO2) for 5 days. In addition to the wells where I co-culture the T cells with the plate cancer cells, I also plate some of the T cells with the activation beads in an empty well to act as a control. I then assess the viability of the T cells by staining with a live dead stain and running flow cytometry. However the results of the flow cytometry show show that most of my T cells are dead (
I am interested in immune evasion pathways and investigating a variety of genes that I have knocked down in skin cancer cells to see if they are important for immune suppression. The objective is to assess how gene knockdown in skin cancer cells affects CD8 T cell functionality. After the co-culture, I stain the CD8 T cells for IFN gamma and granzyme B to see if there any differences in these cytokine staining amounts between the various cancer cell conditions.
The issue is that my T cell only controls (where I culture T cells by themselves in order to see what the baseline T cell cytokine staining patterns are without cancer cells) are mostly all dead by the end of the 5-day co-culture. It could be that I have been doing the co-culture for too long since I read that long culturing with T cell activation Dyna beads could lead to T cell overstimulation and death. I could be also using too little IL2 as I keep the T cell culture media IL2 concentration around 1ng/mL. I have read that others have used IL2 concentrations of 20-30ng/mL. We decided to use only 1ng/mL to be careful not to overstimulate the T cells but this might be a detriment to their viability.
Dear Matthew,
Your work seems interesting. Which type of cancer are you investigating? The results can be dependant on that too. What are the goals of your study?
Sincerely,
Amar, MSc in MLT, Researcher interested in breast cancer
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