PE and FITC stain after fixing cells with 4% PFA - (Jan/27/2014 )

Hey,

Has anyone performed Flow Cytometry staining AFTER fixing the cells?

I have fixed cells that were already labeled with FITC and PE antibodies, but this time I want to label cells only after fixing the cells with 4% PFA. Is it possible?

Cheers,

I presume your targets are surface targets, because if they were intracellular you'd have to fix/permeabilize the cells prior to staining anyway.

You can of course try (wash well after fixing!) and compare the results to when you stained them prior to fixation, but I wouldn't recommend it. When I did that with the surface protein I was investigating, the staining unfortunately got much more positive (we thought it's probably due to the intracellular amount of protein being stained additionally; even if primarily a surface protein, it might be in the Golgi for instance). So in our case we could not stain after fixing. Is it really necessary for you to fix prior to staining ? What exactly do you want to do ?

Thanks for your input Tabaluga.

I want to fix cells first and stain with antibodies later because I am not sure which antibodies I want to use (our cytometer is often offline with tech issues).

There is nothing like testing it, and compare results I suppose.

I have one question tho, If I fix with 4%PFA, the cells become permeabilized (I thought I would need to add a detergent like Triton X100)?

BioPe on Mon Jan 27 15:41:39 2014 said:

I have one question tho, If I fix with 4%PFA, the cells become permeabilized (I thought I would need to add a detergent like Triton X100)?

As far as I know this is a matter of debate, if you browse the internet for this question you'll find that everyone seems to report something different and it probably depends on the time, percentage and temperature...