I've seen a few different recipes for staining/washing buffers used in flow cytometry of human PBMCs and neutrophils, including PBS with 2% BSA and PBS with 0.5% BSA and 0.03% NaN3. We fix our cells first within two hours of patient collection, then stain with our antibodies.
Thanks for your help!
2% BSA with PBS would work just fine. Typically the lower BSA is utilized for cell sorting you need something that won’t bubble on the machine. Since your samples are already fixed they are pretty open to whatever buffer. People typically steer away from FBS only because of the potential for it to activate cells in culture but for flow this doesn’t seem to really be an issue. BSA and FBS is all just protein to help avoid non-specific noise. Either is fine I have not really experienced any inherent advantage over one versus the other besides maybe price.
You can even stain fixed cells in regular PBS. You may consider to add an Fc blocker to avoid not specific binding.
Regular PBS should be fine. To reduce unspecific binding 0.5% BSA may be helpful. But you should be careful fixing your cells before staining. Fixation may destroy antigens and/or you may also get an intracellular staining.
When samples are centrifuged (5 min at 540g) and the cell pellet is washed twice with 5mL of phosphate-buffered saline (PBS; pH = 7.4). Finally, cells we resuspended
in 0.5 mL of washing buffer (PBS + 0.1% of NaN3 (sodium azide) + 0.5% of BSA
(fetal bovine serum)).No problems in most of cases!
I use 2% FBS/PBS. We had better luck using FBS as an Fc blocker than BSA, but both worked just fine with the machine.
If you stain first (in whole blood) you do not need to add protein such as BSA to prevent non-specific binding because the plasma proteins - including immunoglobulins (which would competatively block Fc binding) - are present. You can dilute the sample (say, 100 ul) by adding an equal volume of wash buffer (some PBS/azide based solution, such as BD's CellWash or your in-house buffer) for staining, but it is not necessary. You then lyse (e.g. BD's FACSLyse) then wash, then resuspend in Becton Dickinson's CellFix and leave in the dark (minimum of 30 min - can be overnight or longer - at 4C) and resuspend before acquiring the sample. This "lyse-wash" method is by far the easiest and least prone to "background" uptake of your staining antibodies, but you need to stain when the samples are fresh. You can delay the sample acquisition to suit lab routines.
We foermerly used normal PBS, with excellent results, but we have moved to Becton&Dikinson Facs Flow for more convenience. It is manufactured in 20L containers. We use it instead of PBS for preparing the samples, and the results are optimal (and we use a Beckman Coulter cytometer).
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