Amount of fixation buffer - is it dependent on the number of cells? (Mar/28/2010 )
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
NemaToStella on Mar 28 2010, 11:20 AM said:
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?
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.