Before surface and intracellular staining, should I choose which solution to wash cells? Can I use PBS?
In FACS buffer, what is quantity of FBS? Is the bigger the quantity, the better?
Staining buffer is the buffer used during the staining and it varies according to the step: extracellular (Facs Buffer), intracellular (perm buffer/Facs buffer/PBS) or viability dye (PBS without any protein inside). Facs buffer is usually used to stain extracellularly but it is generally speaking the buffer that you use to resuspend your cells before and after the staining. Usually Facs Buffer is PBS 1%BSA (or 4%FCS) 0,05% Sodium Azide.
For any kind of study whether surface or intracellular staining cells should be washed in PBS. During the staining process we should use FACS buffer containing 2-3 % of BSA in PBS or 5% of FBS in PBS. Due to presence of BSA or other proteins of FBS cells get stable and less sensitive to damage.
Hi Chen Xin Jun
I think the two solutions are the same as they contain BSA at 0.5% or more in buffer saline solution or PBS 1X. Instead BSA you could use other animal serum protein like FBS. These solutions help you to minimize non-specific binding of antibodies.
Instead staining buffer I use buffer saline solution either for cells surface or intracellular staining.
I hope this help you
I always wash my cells in PBS (lacking calcium and magnesium) when preparing them for any type of FACS prep, whether the cells are alive or fixed. The IFA buffer I use for FACS contains 4% FBS.
We use FACS buffer for both washing and staining of the cells (i.e. we prepare our antibody dilutions in FACS buffer). Our FACS buffer is based on PBS and contains 2% FCS, 0.05% Sodium Azide.
We use this buffer for surface staining as well as for intracellular staining.
Staining buffer is the buffer used during the staining and it varies according to the step: extracellular (Facs Buffer), intracellular (perm buffer/Facs buffer/PBS) or viability dye (PBS without any protein inside). Facs buffer is usually used to stain extracellularly but it is generally speaking the buffer that you use to resuspend your cells before and after the staining. Usually Facs Buffer is PBS 1%BSA (or 4%FCS) 0,05% Sodium Azide.
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