After washing in buffer, the cells were pelleted by centrifugation, embedded in 10% gelatin, cooled on ice and cut into 1 mm blocks.
One millilitre of FACS buffer was added and cells were pelleted.
For flowcytometric analysis, CD3+ were kept on ice and washed extensively with citrated PBS containing 1% FCS. 10 µl of FITC, APC or PE- conjugated antibody was added and incubated on ice for 20 min. 300 µl FACS buffer was added and T cells were pelleted, resuspended in 200 µl buffer.
The cells were incubated again on ice for 15 minutes, 10 times the labelling volume of depletion buffer was added, and the cells were pelleted by centrifugation at 300 g for 10 minutes.
Cells were pelleted, resuspended in buffer A (20 mM Na acetate, pH 5.1, 100 mM NaCl, and 1 mM EDTA), and lysed by passage through a French press or high-pressure homogenizer.
Stained cells were pelleted and resuspended in buffer containing DAPI to exclude dead cells.
Remaining GFP+ xenograft derived cells were pelleted and resuspended in lysis buffer for western blot analysis.
Cells were pelleted and resuspended in HB buffer.
Cells were pelleted, incubated and resuspended in NP-40 lysis buffer with protease inhibitor cocktail.
Briefly, cells were pelleted, supernatant removed and pellet resuspended in RLT lysis buffer.
