Most of the standart protocols for staining (direct or indirect) require you to wash cell suspension after every addition of the antibody or fluorescent dye.
But, do we really need this step? For example, if Iam staining my cell suspension, that has a volume of 20 uL, with some dye conjugated antibodies. And then, instead of washing the cells, I just increase the volume of the suspension to 200 uL with staining buffer. As I understand, in this way the concentration of the free antibodies in solution will be sufficiently low. And at the same time, flow cytometer will detect for the most part only cells, with SSC and FSC.
So, is it ok to do so, or there is something I've missed, and we really should always wash our cell suspension from free antibodies?
It is not absolutely necessary to wash cells after staining if you are using directly conjugated antibodies, but not washing the cells can lead to higher backgrounds. If you are using secondary stained antibodies, they can form immune complexes with the unbound primary antibodies, and stick non-specifically to cells.
One assay where washing cells is contraindicated is annexin-V:PI staining. I never wash the cells after incubating with annexin-V and PI; I dilute them with binding buffer and run them on the flow cytometer.
Trypsinized cells actively perform endocytosis and macropinocytosis that can take up the dye resulting in non-specific background increase. it may not have a major issue if you have one or two samples and the flow runs quickly. If you have more samples it increases flow time thus giving more macropinocytosis.
washing helps removal of unbound antibodies, so that the second fluorescent antibody can efficiently label primary antibody.
Thank you very much for your answers! Goodwin G. Jinesh, Tanisha Singh, Gary Lee Gilmore
The old adage "That depends" definitely applies to whether or not to add isotype controls. If your cells of interest have Fc receptors, then either isotype controls or FcR-blocking antibodies are essential - isotype controls are a way to document that.
Another aspect where isotype controls may reveal something of interest can occur when you are testing different sources of cells - in this case, different strains of mice - where the isotype control one group had a high background on one strain [C57Bl/6] but not on a B6 congenic strain. The isotype control they were using was an anti-NP monoclonal antibody [IgG1,kappa] from BALB/c mice, and the congenic strain carried the BALB/c allele of a minor histocompatibility antigen known as H8. This indicated that the B6 H8 allele serves as an auto-antigen that deletes out the anti-NP kappa responses that BALB/c mice make, but C57BL/6 do not. Their anti-NP antibodies have lambda light chains.
So isotype controls have their uses.
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