Im doing flow cytometry using 8 different antibodies. I have to optimize the protocol before i run my final samples. I have to use zombie UV which requires PBS (no serum) washing of cells before addition following by antibodies.
However, recently i got non specific binding of cells and have to Fc block. Fc block is prepared in stain buffer which contains serum. my sequence of adding these components are as follow:
Fc block--> zombie UV--> antibodies
in Fc block, the protocol says no need of washing required, add antibodies directly.
however, Fc block was prepared using stain buffer which has serum, and before adding zombie UV i need to wash with PBS so no residues of serum remain.
my question: is centrifuging Fc block enough? or should I wash the cells with PBS before adding zombie UV? what if washing will make Fc block ineffective?
2. Can I use 7AAD dye in the end of protocol instead of zombie UV?
Hi Shali,
Given the conditions of UV zombie, I would highly recommend washing your cells after FcBlocking to make sure there are no rests of serum in your sample that can bind to UV zombie and create a non-specific signal. Most viability dyes interfere with serum. In my experiments, where cells come from a culture, I wash them twice before viability staining. Hence I would advise you to wash your cells with PBS at least once before proceeding with antibody staining.
I can not help you with question 2 though, sorry and I hope this come in hand helpful!
If your fear is having a weak signal, maybe you could try increasing the dilution or the incubation volume, but I would not skip the washing step, as avoiding it can lead to non-specific binding due to serum presence, even though you use Fc blockade.
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