I'm used to PBS 2% FCS. FCS should block aspecific binding of the antibody, so I'd say the second option is the one you want.
Hello Justine,
I use PBS without serum since almost 20 years, and I do not have any problems with unspecific binding.We analyse mainly cells in whole blood as well as cultured cells such as MSC. Also if analysing cultured cells we do not see differences between buffer supplemented with FCS respectively buffer without FCS. Maybe the FCS in the culture medium provided a sufficient blocking.
Good luck for your experiments
Dirk
My lab typically does flow cytometry staining of mouse lymphocytes with 0.5% BSA in PBS. However, we wash with PBS once or twice prior to CFSE loading, intracellular staining, or fix-and-perm, and also do all of these in PBS only. When CFSE loading is done we "quench" with FCS.
Our lab also stains in PBS-0.5% FBS for surface markers. Especially for primary cells, the FBS helps preserve viability.
Thank you for all your input. I was thinking the same thing about the 2% FCS reducing nonspecific binding, but was wondering since others use only HBSS if there was another advantage to HBSS alone that I might not know about.
You should use 2% FBS or 0.5% BSA. It helps maintain cell viability. For preventing non-specific antibody binding, you could use Fc-receptor blockade reagent or simple RAT serum. Fc-receptor block is highly recommended if you are staining Macrophages, DCs, B cells and eosinophils. For T cells you don't necessarily need add, unless you want.
We normally used PBS 2% FCS, but in some occasion the presence of FCS induced non-specific binding of our antibodies. FCS is also normally recommended if you want to keep your cells alive and healthy (ie post-sort), but according to my own experiment I would say try both, make the good controls (with or without FCS, different antibodies targeted at the same molecule (ie rabbit anti-mouse/human, goat anti-mouse/human... whatever is available, different fluorophores coupled to the same antibody (same clone)) and then use whatever is best for your own experiment.
Normally the fbs/bsa act as a cushion to protect the cells from breaking during the various passages when you prepare the cell solution for flow cytometry and keeps them perfectly viable if you want to seed them later. I would say when i do it on live blood cells i always use fbs/bsa because they are more delicate, if i am fixing them i don't use fbs. As a general rule, unless fbs changes your results (eg antibodies biding) use it all the time is a neutral substance for flow so i don't see why you shouldn't
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