Hi Reza,

One reason why that might slighlty matter could be that the fluorescent conjugates might be affected by the ph difference when the lysis buffer is added. So it also depends a little on how you treat your compensation controls as well as staining controls. We always do RBC lysis before staining ( so yes, you definitely can), that way we eliminate the RBCs directly from the beginning which then don't interfere with staining at all. But as also mentioned above, both approaches are very common.

I’ve always stained after the RBCL step. Wash your cells after RBCL application with antibody binding buffer and proceed. However, you may want to look into the history of your current protocol development. It may be that your target cells work better in the binding reaction in the presence of blood etc. Find out why the protocol is the way it is, you may be overlooking something.

Hi Reza,

I think that the LSW sequence would work better because;

1) with this sequence chance of binding your Ab to RBCs will be decreased

2) the lysis step does not affect the efficiency of staining procedure