I am making hypoxia situation in human pigment epithelial (RPE)using CoCl2.
I read the some paper that the cobalt could make hypoxia situation and reduce the cell viability.
however i used from 50 to 500uM in the serum free medium for 24h,
the viability is increased. The viability is measured by CCK-9
What do I wrong?
My RPE is passage #7 and cultured in DMEM with 10% FBS.
Cobalt Chloride ( or hypoxia ) induces HIF1 and it's pro-survival pathways to adapt to hypoxia and upregulate glycolysis, which can explain the better survival you see given that you are starving the cells from serum. You can try hypoxia or other inducers such as DMOG or Nickel Chloride . Have you tried in the presence of serum or longer timepoints 48 , 72 hours ?
Dear Andreas thank you for your kind answer.
I only tried non-serum media with cobalt and also doing 72h.
At 72h, over 200uM of cobalt chloride, the viability of the cell are decreased.
However, under 200uM, all of the case had higher viability.
So you meas the hypoxia enhance the viability of the cell in first time and then cells are going to death?
And should I do in serum containing media?
The papers I read performed the serum-free media and in my opinion the serum free media is better for see the effect of hypoxia.
But I didn't think the starving could affect.
Dear William,
I have used CoCl2 250 uM to induce overexpression of GLUT1 in a cell line called clone9 (hepatocytes from rats), and I Andreas said, this treatment could increase viabilty by preparing the cells to face an hypoxia event and therefore, It ca not be considered as an hypoxic stimulus rather is a inducer of hypoxia adaptation.
For Hypoxia simulation, I suggest you to use: desoxyglucose, sodium azide, shutting down mitochondria with FCCP, for instance. there ar many ways to test.
you will do it. go ahead.
OP
Dear Omar
Thank you for your comment.
You and Andreas give me a great information.
I will check other material for inducing chemical hypoxia.
And also do same exp with serum containing media.
Thanks a lot both of you.
Sincerely
Will
Dear Andreas and Omar, I'm sorry to late report.
I tested 200uM and 500uM of CoCl2 with and without FBS media in my RPE cell.
The results is similar with previous one in without FBS case for 24h and 72h.
The viability (or cell number, I don't know it is right explanation, anyway the CCK-8 signal) is higher at both 200uM and 500uM than control at 24h . At 72h, the 200uM is higher signal than control but 500uM has slightly less signal than control.
In case of serum containing medium, the 200uM and 500uM is less than control in both 24h and 72h cases.
I don't know exact reason of these opposite results (maybe the pathways, you explained, thank you for information i have to study). But now, I understand, what condition I have to use.
Thank you again both of you.
Sincerely
WIll
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