T cells were isolated from peripheral blood mononuclear cells (PBMCs) using a human T cell negative selection kit (STEMCELL, 17951) and cultured in T cell expansion media (Gibco, A1048501) at 37°C with 5% CO2. GCSF or equal volume of vehicle control was added at 10 ng/ml. Stimulation was achieved at 6 hours with 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin. Standard protocol was used to perform staining as we have done previously (23 (link)). Antibodies used in this study were CD4-BV650 (RM4-5) (BDBiosciences, 9122894), CD8-APC-Cy7 (53-6.7) (Biolegend, B283110), and IFNy-BV421 (XMG1.2) (BDBiosciences, 8144795). Cells were run on a Stratedigm S1400Exi flow cytometer. Data was analyzed using FlowJo V10.8 (FlowJo, RRID:SCR_008520).
Protocol detail
T cell Activation and GCSF Treatment
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Antibodies
Cells
Cultured media
Gcsf
Human
Ionomycin
Peripheral blood mononuclear cells
Phorbol myristate acetate
Stemcell
T cell
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Variable analysis
independent variables
- GCSF concentration (10 ng/ml)
- IFNy expression in CD4+ and CD8+ T cells
- T cell culture conditions (37°C, 5% CO2)
- T cell expansion media
- Stimulation with PMA (50 ng/ml) and ionomycin (1 μg/ml)
- Staining protocol and antibodies used
- Positive control: T cells stimulated with PMA and ionomycin
- Negative control: T cells treated with equal volume of vehicle instead of GCSF
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