OT-1 Cell-Mediated Cytotoxicity Assay

Splenocytes (10⁶/mL) from OT1 mice were stimulated in RPMI + 10% FCS with 1mM OVA peptide for 3 days, washed and cultured with IL2 as above for 3 more days. Targets EL-4 cells loaded with Cell Trace Violet (C34557) at 2uM in PBS for 10 minutes then quenched and washed. Cells were pulsed with 1uM SIINFEKL peptide for 30-60 minutes at 37 degrees. Target cells were incubated with activated OT-1 cells, at varying ratios (1:5, 1:10, 1:20 in 200 mL media for 4 hours. Stained w/APC-anti-CD8 and live/dead for 30 minutes on ice. Acquired on Beckman CytoFLEX cytometer.

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Tarasenko T.N., Banerjee P., Gomez-Rodriguez J., Gildea D., Zhang S., Wolfsberg T., Jenkins L.M, & McGuire P.J. (2023). Pyruvate dehydrogenase complex integrates the metabolome and epigenome in memory T cell differentiation in vitro. Research Square.

Publication Preprint 2023

CytoFLEX cytometer

Cells Mice Peptide Siinfekl peptide Violet

Corresponding Organization : National Institutes of Health

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Variable analysis

independent variables

Ratio of activated OT-1 cells to target cells (1:5, 1:10, 1:20)

dependent variables

Cytotoxicity of activated OT-1 cells against target cells

control variables

Splenocytes from OT1 mice
Stimulation with 1mM OVA peptide for 3 days
Culture with IL2 for 3 more days
Target EL-4 cells loaded with Cell Trace Violet at 2uM
Pulsing target cells with 1uM SIINFEKL peptide for 30-60 minutes at 37 degrees
Incubation time of 4 hours
Staining with APC-anti-CD8 and live/dead marker
Acquisition on Beckman CytoFLEX cytometer

positive control

Not mentioned

negative control

Not mentioned

Annotations

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