Cell fixation - (Nov/18/2004 )
How Paraformaldehyde (PFA 0.5%) "fixes"the cell before a flow cytometric analysis??????
I'm not sure what the question is, but PFA works by crosslinking proteins, and thus fixes the cells. You can fix cells before FACS, as it's easier to handle and you can leave them in the fridge etc.
Regarding cell fixation, I've been having problems trying to detect beta1 integrin in an epithelial cell-line by immunofluorescence. In your experience, does fixation with 0.2% paraformaldehyde for 10 mins at RT alter antigenic epitopes, especially for membrane proteins?
Thanks for your insight.
well, that depends on the antibody. Some antibodies works best with methanol fixation, some with PFA. This is usually specified in the spec-sheet it's a commercial antibody, otherwise look up previous publications using that specific antibody, or try different combinations yourself. Another problem could be whether the epitope is intra- or extracellular. If intracellular, you also need to find the optimal permeabilising agent (methanol, TX100, saponin, etc).
Thanks all of you for your interest in my question.
I stain cells with monoclonal antibody cell surface markers and analyze them by using flow cytometry. Before any procedure, the protocol specifies that we should use PFA 0.5% to "fix" the cell.
My specific question was how does PFA fix the cell? Is the protein crosslinking still a possible answer for this?
My name is Ana. And I´m having a similar problem.
Briefly: I work with T cells, I label them and then fix with 1% PFA in PBS, 5min, RT. Usually I only do fixation when I have to use the FACS the next day (as I don't have one in my lab) or when I use the cells for confocal microscopy.
The problems is that when I don't fix the cells I can see a good percentage of expression of my protein (a membrane receptor) but after fixing the positive percentage "disappears" . So I see the positives as negatives.
Then I think the problem is that the praformaldehyde somehow destroys my epitope, or (as i have already labeled the cells) or the union between antibody-protein.
I am looking for a new fixation method, could you recommend me one? or is there any tip to avoid this problem?
Thanks to all
PD: no, the manufacturer says nothing about the fixation method. Its scientifc support still recommends paraformaldehyde.
Regarding to the fixative 4% paraformaldehyde for FACS analysis, I have two questions:
1. Some said that after i made 4% paraformaldehyde, aliquot them into small volumes, then i can store them into -20 degree C. before use, thaw a certain amount of 4% paraformaldehyde and after use put them into 4 degree C and it can be usable in 5 days. My question is after thawed, can I re-freeze them into
-20 degree C freezer for longer storage?
2. after i incubate the cells into 4% paraformaldehyde, can I directly leave the cells in 4% paraformaldehyde overnight or longer until i run the cells onto the FACS machine?
Thanks for any comments and advices!