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.