Hello Tayebeh.

When cells are frozen and thawed lots of cells lyse and rupture without cryoprotectant and appropriate freezing procedure. The the released cytoplasmic proteins and varying sizes of vesicular membranes generated from the ruptured cells would hinder accurate separation. I suggest fixing cells in 4% PFA for 20 min, washing the excessive PFA by spinning down 3 times in 1.5ml Eppendorf tubes, transferring the cells into 1% (w/v) BSA/ml buffer, and keeping the cells at 4C to prevent cell aggregation caused by the fixative until use. For staining, block the aldehydes on the cells by incubating cells in 0.26% NH4Cl solution for 20 min. The remaining procedures are same as in routine FACS.

Regards.