This protocol uses BD Bioscience intracellular kit (Cat # 555028)
- Cell harvest/ Stimulation:
TIL: Harvest 10mm tumor and process through metal screen. Pass cells through a 100um filter followed by 40um filter and resuspend in 5ml M. Plate 1ml in a 24 well plate and add 1ul peptide. For PMA/iono add 2ul PMA (50uM) and 5ul Ionomycin (100uM).
Splenoctyes: Harvest spleens and plate 1x106 cells/well (in 100ul M) in a 96 well flat bottom plate. Stimulate with a 1:1000 dilution of peptide. For PMA/iono add 2ul 50uM PMA/ml and 5ul 100um Iono/ml.
T cell clone: Plate 5x105 T cells/well in a 96 well flat bottom plate with 1x106 splenocytes and 1:1000 peptide (in 100ul M total). PMA/iono same as above
Stimulate cells for 1hr at 37˚C.
- Add equal volume of golgistop diluted 1:750 in M (1ml for TIL, 100ul for others). Incubate for an additional 3-4hr.
- Harvest cells and transfer to a round bottom 96 well plate (For TIL transfer 1/2 of a 24 well into each 96 well).
- Spin down cells and stain for cell surface markers in 50ul FCWB for 30 min at 4˚C.
BE SURE TO ADD Fc BLOCK TO TIL SAMPLES
- Wash cells 2x with FCWB and add 50ul Cytofix/Cytoperm to each well. Incubate 20 min at 4˚C.
- Wash cells 2x with 1x Perm/Wash. Stain for intracellular cytokines in 50ul Perm/Wash for 60 min at 4˚C. (Overnight may be better and doesn’t cause too much background)
- Wash cells 4x with Perm/Wash and 1x with FCWB and analyze by flow cytometry.