Hello Ahmed Al-Omar

You should fix the cells in 4% PFA for 10-15 mins after isolation. Then wash the cells in PBS, which is important, and resuspend the cells in PBS/BSA solution which should not have any fixative. Then you can store the fixed cells at

4 degree C and carry out staining after 24 hours. Do not extend beyond 48 hours. During transit, you can transfer both the cells and antibodies on ice.

Best.

Malcolm Nobre is it fine to store the cells without fixing them in 4% PFA? I only have absolute methanol can I use it instead? If I can how to dilute it and prepare it as a fixation solution?

Thank you

Dear Ahmed Al-Omar

I would suggest you fix your cells and I recommend using 4% PFA.

You can use methanol for fixing. The protocol is as follows:

Suspend the cells in 700-900 μL of ice cold methanol. Incubate for 15-20 minutes at 4 degree C. Centrifuge the cells for 5 minutes at 1250-1500 rpm. Remove and discard the supernatant. Wash 2 times with PBS. Store cells in PBS at 4 degree C before staining.

Methanol fixation can also permeabilize the cells and is not always suitable. Methanol fixation might decrease the availability of surface antigens. It can also cause loss of signal. So, it is better to use 4%PFA instead.

Best.

Malcolm Nobre Thank you for your recommendations that were helpful.

Sincerely

If it’s less than 24 hours, you can leave them unfixed. But then you have to account for cell death. Better to fix them.