2 replies to this topic

[NemaToStella]

Hi,
this may very likely be a stupid question, but I'm going to ask anyways:
for my ChIP experiments I often get quite different numbers of cells (as they're from dissociated tissues I have no control over the number of cells I get), but so far I've fixed in the same volume of fixation buffer (5 ml, 1% formaldehyde). In your opinion, should I increase the volume of the buffer (of course keeping the%formaldehyde constant)? Or is n(formaldehyde) not the limiting factor? I'm just concerned I'm not fixing individual cells enough if there are too many.
I'd appreciate any thought on that.

NemaToStella

[KPDE]

NemaToStella, on Mar 28 2010, 11:20 AM, said:

Hi,
this may very likely be a stupid question, but I'm going to ask anyways:
for my ChIP experiments I often get quite different numbers of cells (as they're from dissociated tissues I have no control over the number of cells I get), but so far I've fixed in the same volume of fixation buffer (5 ml, 1% formaldehyde). In your opinion, should I increase the volume of the buffer (of course keeping the%formaldehyde constant)? Or is n(formaldehyde) not the limiting factor? I'm just concerned I'm not fixing individual cells enough if there are too many.
I'd appreciate any thought on that.

NemaToStella

I guess you could quench the formaldehyde if you had a large enough amount of cells but I doubt you've got that many.  
Whats the upper limit for the number of cells you get?

[NemaToStella]

Thanks for the fast reply, KPDE!
I'm guessing I have around 10E8 cells, I have never counted though. Just to be sure I'm now fixing the cells in 25 ml.