FACS 5%FBS vs 1%BSA vs FACS Buffer - (Feb/26/2008 )
I've been using 1xDPBS to resuspend my cells for FACS analysis, but lately I've been getting bad results and my boss suggested that I used 1%BSA. I have also seen some protocols where they used 5% FBS and other protocols that just call for FACS Buffer. What is the difference in using 1% BSA vs 5% FBS and what exactly is FACS Buffer composed of? Thanks.
what exactly are you trying to do??
if you are just doing FACS analysis, cell is already fixed, so FBS doesnt do anything (at lease I never use FBS)
are you sorting cell?, if so I normally use small amount FBS, BSA or FBS should not be matter, it just makes cell happy until all the cell will be sorted.
If you are planning to culture the cells after FACS, might be better to have them in 5% FBS to keep them happy.
the cell are fixed in 4% paraformaldehyde and i'm only using them for analysis then i toss them afterwards..
okay so thanks for the info.. much appreciated =)
FACS Buffer we use has 1% BSA and 0.1% Sodium Azide.
No one mentioned of Sodium Azide here (of course not if U want to culture later). Is it not commonly used? Fixed cells also will not need Sodium Azide but is it necessary to render cells metabolically inert before fixing by exposing to Sodium Azide or not?
If the cells are fixed then what will it make difference if it is BSA or FBS? Pumpkin21, please let us know if U see any difference. I can't think of any.
when we do Facs analysis (no culture later), I use Na Azide (in PBS) to fix them, and later I washed and reconstitute with 1% formaldehyde, I think
BSA and FBS (or any other serum for that matter) will accomplish pretty much the same thing when staining cells for flow cytometry. there is no need to use sodium azide in these buffers, it will only hurt your cells. the purpose of the azide in these buffers is to prevent microbial growth, but these buffers are used so quickly (and are extremely cheap to make) that you shouldn't run into any problems. another reason that people use 'protein containing buffers' for flow cytometry is to prevent cells from sticking to the side of plastic tubes (or other culture labware) as well as preventing cell clumping.
ps. you don't need to use such a high percentage (5%) of fbs unless you are sorting. using as little as 0.5% is fine.