Phenotype analysis of PigA-deficient mast cells by flow cytometry. (A) PigA-deficient BMMCs cultured from VavCrePigALoxP (KO; bold lines) or littermate control VavCre (WT; filled lines) mice were analyzed by flow cytometry. Complete GPI-deficient phenotype was evident from a lack of staining with proaerolysin, a general marker for the GPI lipid anchor, and with antibodies against the GPI-AP CD48 and HSA. PigA-deficient BMMCs demonstrated normal staining with antibodies against the mast cell marker c-Kit, and with the lipid raft marker cholera toxin subunit B (CTx); and normal binding of monomeric mouse IgE. Dotted lines: staining controls. (B) Peritoneal mast cells from littermate control VavCre or VavCrePigALoxP mice were stained with anti–c-Kit and proaerolysin; c-Kit positive cells were gated in forward/side scatter. Negative proaerolysin staining demonstrates in vivo mast cell GPI-deficiency; this was confirmed using antibodies against CD48 and HSA (not shown).