5 replies to this topic

[Hamster]

Hi there.

I'm trying to induce hypoxia in my cell cultures by placing them in a 2% O2 incubator for over 24hours. I'm wondering if there's a common practice of putting CoCl2 into PBS for the cell washing in order to prevent reoxygenation of cells...and if so what's the concentration of CoCl2 being used? 100uM? I did that but when I tried to extract proteins with the urea buffer a strange precipitation reaction occurred to give a brown precipitate with the lysate I have. I'm kinda worried that this will mess up with my proteins...

Thanks a lot!

Edited by Hamster, 28 June 2010 - 04:35 PM.

[laurequillo]

Hamster, on Jun 29 2010, 02:34 AM, said:

Hi there.

I'm trying to induce hypoxia in my cell cultures by placing them in a 2% O2 incubator for over 24hours. I'm wondering if there's a common practice of putting CoCl2 into PBS for the cell washing in order to prevent reoxygenation of cells...and if so what's the concentration of CoCl2 being used? 100uM? I did that but when I tried to extract proteins with the urea buffer a strange precipitation reaction occurred to give a brown precipitate with the lysate I have. I'm kinda worried that this will mess up with my proteins...

Thanks a lot!

Hi there. Actually I never used CoCl2 and real hipoxia together. I just placed my cells in hte incubator for a given time, using as less medium as possible. And then next day put the plates in ice and do the lysis as fast as possible. I guess you can use your CoCl2, but for me it was no neccesary

[Hamster]

Did you get an obvious induction of HIF1a?
And how many hours did you incubate your cells for? 24hours?
How come less medium?
Have you ever heard of anyone/papers who did put CoCl2 in their washing? I do read about people placing plates on ice but not CoCl2 in PBS...

Sorry lots of questions in one go

laurequillo, on Jun 29 2010, 04:47 AM, said:

Hi there. Actually I never used CoCl2 and real hipoxia together. I just placed my cells in hte incubator for a given time, using as less medium as possible. And then next day put the plates in ice and do the lysis as fast as possible. I guess you can use your CoCl2, but for me it was no neccesary

[laurequillo]

Yeah HIF1a was induced.
You use less medium to improve the hipoxia (if you use too much medium you have more O2 in the medium and then it takes more time to reach the desired O2 levels).
I used to do it overnight
No, I did not use CoCl2 in the PBS, and actually I dont know if people do that.

[Hamster]

http://www.springerl...2840pm80w206wx/

In this paper they've mentioned using PIB in addition to ice cold PBS for washing...What's the use of PIB exactly? Google doesn't seem to help much :-/

Thanks!

[laurequillo]

Sorry i did not read your reply. I will check the paper and i will let you know