Cell Fixation for Long Storage Time

[Dionisia]

Hi,
Im working with mouse cells and i need a quick method to fix these cells and keep them for later analysis.
It will be mainly surface and intracellular antigens using flow cytometry.


Is there a good method for these types of samples and how long can we keep these samples before we label them?

What is also the best method for permeabilising these cells?
thanks

[ethidiumbromide]

When you say "cells", do you mean a tissue sample? or do you mean actual cells? And when you say "fix" what do you mean exactly? Freeze? Fix, as in fixing cells to a slide?

If cells, I think you should be able to freeze them -80 in cell culture media with 10% DMSO. If tissue, I'm not sure, but maybe just like it is?

When we fix our cells, we use 1-2% paraformaldehyde for 5-10, then wash 2-3 times with PBS. When we permeabilize cells we use 0.1% Triton X-100 in PBS for 2 minutes and then wash with PBS 2-3 times. But this protocol is for IF. I don't know if it would be different for flow.

If you can be more specific, I can ask someone in my lab on Monday and try to get you a protocol. We do flow all the time. Just not by me.

[Levitron]

Hi,

I'm culturing cortical neurons and I'm looking for a protocol to store PFA fixed cells so that I don't have to stain them all in one go. How long can you store PFA fixed cells?

Thanks a lot for your help!!!

[airie]

Hi I read somewhere that if your antigens are on the cell surface it might not be very good for the immuno if you permeabilize your cells....if i understand correctly to permeabilize means to poke holes in the membrane so the antibody can access maybe intracellular antigens but making holes in the membrane might cause the loss of the actual antigens?

[haisond]

Levitron,

Are the fixed cells attached or suspended? For attached cells fixed onto plate wells, I've used a cryoprotectant solution as described in the cryoprotectant recipe from IHC World. Depending on your antigen, this can preserve some antigens for up to a year at -20C. Works for storage of brain slices as well. For fixed suspended cells, used 10% DMSO in HBSS or Hepes.

best,