cobalt chloride not inducing hypoxia - (Jul/28/2012 )

hello friends..
I am trying a Hypoxia induction on PC 12 cells by using CoCl2 i have read certain papers n concluded that 150-200uM conc. is enough for 40-50% cell death..But when i m trying this in my lab there is no Hypoxia even at 300uM conc....
earlier i thought there is some problem in my calculations or my stock so i prepared the stock again but still the result is same.. what do i do???plz help anyone???its kind of frustrating....
i am giving a 24hrs incubation n cells are maintained in 5% serum and evaluation is done by MTT assay(we do cell synchronization also)...
plz help ..anyone...

I'm not an expert on cell culture, but PC 12 is a pheochromocytoma cell line that has alterations in hypoxic pathway. Maybe these cells behave differently. You need to find papers about your specific cell line.
I found a paper using cobalt on PC 12 and their viability chart have 50 % cell death in concentrations around 600 uM of CoCl2.
It's not freely accessible so I send it to you as a PM.

J Neurosci Res. 2001 Jun 15;64(6):646-53.
Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation.
Zou W, Yan M, Xu W, Huo H, Sun L, Zheng Z, Liu X.

I also note that you don't appear to be detecting the hypoxia directly or indirectly - so how do you know that they aren't hypoxic, just not enough to kill them?

Look at this paper: Up-regulation of hypoxia inducible factor-1 alpha by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells.

Journal of Experimental & Clinical Cancer Research 2012, 31:28. 

They use PC cells and CoCl2 concentrations from 50-200 um/L and incubation times from 24 to 120 h. Cells don't show hypoxic signals until 96h in this cell type.

Hope it helps