MITOGENIC ASSAY FOR 32-D CELLS
1. SETUP ASSAY
Grow up cell lines (include positive and negative controls!!); you will need about 107 cells per line. Count cells, and take 107 cells of each line, spin down and wash twice in RPMI + FBS + P/S (no WEHI) [MITO MEDIA]. Resuspend to a final concentration of 2 X 106 cells/ml in same media.
Setup one 24-well plate per cell line: each well should contain 1 ml of media. I use 3 wells per condition and use a) MITO media, b) MITO + 10-10 M insulin, c) + 3 x 10-10, d) 10-9, e) 3 x 10-9, f) 10-9, g) 3 x 10-9, h) 10-8, i) 10-7, j) WEHI (normal medium).
Put 100 ml of cells into each well. Return plates to incubator for 48 hours (-ish).
2. LABEL AND HARVEST
Dilute 3H-thymidine 1:10 with MITO media. Add 20 ml per well of mitogenic assay plates. Return cells to incubator for 3 hours.
Remove cells from incubator. Collect cells and remove unincorporated nucleotide: using a transfer pipette, squirt cells onto glass microfiber filter on vacuum apparatus. Wash filter 3 times with water. Transfer filters to scintillation vials, allow several hours to dry, add scintillation fluid, and count.