How to determine the cytotoxic effect of CAR T cells on lymphocyte B cell line?

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Costanza Maria Cristiani

Hi Maja!

If you want to check the capability of CAR-T cells on B cells (or any other combination of effectors and targets), I would recommend the LDH Assay. It is a colorimetric assay based on LDH release by dead cells, very simple and reliable. I use the kit from Roche, but similar kits are provided by many companies. I hope it helps!

Malcolm Nobre

Hello Maja Soltyka

Please refer to the articles I have attached below for your reference. They will be helpful.

Article Comparative analysis of assays to measure CAR T-cell-mediate...

Chapter Determination of Cytotoxic Potential of CAR-T Cells in Co-cu...

Best.

Ahmad Al Khraisat

The cytotoxic effect of CAR T cells on a B-cell lymphocyte line can be determined by performing a standard cytotoxicity assay such as the lactate dehydrogenase (LDH) release assay or a flow cytometry-based assay.

Lactate Dehydrogenase (LDH) Release Assay:

Seed the B-cell lymphocyte line in a 96-well plate and allow it to grow to confluency.

Add CAR T cells to the plate at different effector to target (E:T) ratios (e.g. 1:1, 5:1, 10:1) and incubate for a certain period (e.g. 24-72 hours).

Collect the supernatant from each well and measure the amount of LDH released using a commercial LDH assay kit.

Calculate the percentage of LDH release from each well relative to the maximum LDH release (obtained by adding lysis buffer to the B-cell lymphocyte line).

Plot the data as a graph of percentage of specific lysis versus E:T ratio.

Flow Cytometry-based Assay:

Label the B-cell lymphocyte line with a fluorescent dye such as CFSE.

Seed the labeled cells in a 96-well plate and allow them to grow to confluency.

Add CAR T cells to the plate at different E:T ratios and incubate for a certain period.

Harvest the cells from each well and stain them with a viability dye and a marker specific to the B-cell lymphocyte line (such as CD19).

Analyze the cells by flow cytometry to determine the percentage of viable B-cell lymphocyte line cells remaining relative to the initial number of cells.

Both methods can be used to determine the cytotoxic effect of CAR T cells on a B-cell lymphocyte line. The LDH release assay measures the amount of LDH released by damaged cells, while the flow cytometry-based assay measures the percentage of viable cells remaining after exposure to CAR T cells.