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Cell fixation


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4 replies to this topic

#1 thegradstudent

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Posted 18 November 2004 - 04:23 AM

Hi!

How Paraformaldehyde (PFA 0.5%) "fixes"the cell before a flow cytometric analysis??????
:)
TheGradStudent

#2 Sprag

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Posted 19 November 2004 - 07:36 AM

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.

#3 Newbie

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Posted 29 November 2004 - 06:32 AM

Hi,

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.

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#4 Sprag

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Posted 07 December 2004 - 08:49 AM

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).

good luck,

#5 thegradstudent

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Posted 07 December 2004 - 02:57 PM

Hi!
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?

Thanks
TheGradStudent




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