A.  The cells grow in RPMI 1640 supplemented with 10% FBS, 5% WEHI conditioned media and Pen/Strep solution:  Use Sigma hybri-max P/S solution and add 1% to GIBCO media (which is unsupplemented) or 0.5% to Joslin TCC media.  For IRS-1-expressing cell lines (or cell lines expressing other pCMVhis expressed proteins), add 5-10 mM histidinol to the media.


Thaw the cells in 37 C water bath (no shaking).  If only a few cells survive its OK:  they will come back.  They grow fast.  Once at sufficient density, they double every 12-14 hours (estimate).  They will die at densities >= 2 x 106/ml. 


They are suspension cells.  Grow them in flasks and split by resuspending settled and quasi-attached cells and transferring some to a new flask with new media.


Spin cells at 1500-2000 rpm in table top centrifuge (15 or 50 ml conical tubes) 3-5 minutes.


B.  Freeze log-growth cells by spinning down 10-20 mls of cells and freezing 3 x  1.5 ml cryovials in RPMI + 20% FBS + 5% WEHI + 8% DMSO.  Freeze -20 C 1 hour, -70 C overnight and transfer to liquid nitrogen..


C.  Use lots of cells per experimental point.  For blotting lysates you want at least 5 x 106 cells.  For IP's or enzyme assays use 5 x 107 cells per point (This number is 1 large confluent flask with 50 ml of media).


D.  Typical experimental protocol:  Spin cells down in the AM (early) 1 point = 1 flask= 1 50 ml conical tube.  Resuspend the pellet in each tube in unsupplemented DMEM and return tubes (caps loose) to incubator for 4-6 hour step down period.  Cells will settle out, thus tubes should be swirled every 30 minutes to resuspend.  Stimulate with desired reagent desired amount of time, fill 50 ml tube with ice-cold PBS and spin cells out 3-5 minutes, refrigerated.  Pour out PBS/DMEM and lyse cells....

NOTE:  JHP amplifies signal by pretreating with VO4 (50 mM, 30 minutes) and includes same in PBS for cell collection following stimulation.  I don't do this, as it raises background in IRS-1 alone cells.