Protocol Online logo
Top : New Forum Archives (2009-): : Flow Cytometry

Question about fixation and wash - (Oct/02/2012 )

I have a problem about flow sample preparation. We fix A549 cells with 0.5% PAF for 1 h in 4degC. At the end of fixation, cells were centrifuged with 1200 rpm, 5min, then washed with PBS and centrifuged with same speed. We found that cells were seriously lost (cannot precipitate) after second centrifugation. Can everyone tell us how to change our condition for avoiding sample lose? thanks.

-weining-

Have you tried a higher speed spin?

Of note - if you are talking about centrifuges, rpm is not helpful as the force applied by the centrifuge is dependent on the radius of the rotor - you need to refer to RCF or gravity ("g") forces

-bob1-

Are you fixing the samples just prior to FACS acquisition? In that case, you can just remove the antibody mix with one spin and then resuspend the cells in the fixative and proceed straight to the acquisition stage. Avoiding the extra spin steps can prevent the loss of cells.

In case you are fixing the cells and have to really do all the spins, try going for 300G for 10 minutes. Once fixed, it is easy to lose cells and so a longer spin time might get them pelleted. I never go lower than 1500 rpm and 6-7 minutes even when I am working with cells that are not fixed.

-zodiac1505-