Example 1
B Lymphocytes were Purified from Peripheral Blood Mononuclear Cells (PBMCs) obtained on a Ficoll® gradient after removal of the T lymphocytes (rosette technique using sheep red blood cells pretreated with neuraminidase) and of the monocytes (negative depletion technique, B cell kit without CD43, STEMCELL TECHNOLOGIES™). The purity of the CD19-positive LBs was verified by flow cytometry showing a purity greater than 95%.
A/B—Protein analysis of the LBs by Western blotting on SDS-PAGE made it possible to distinguish, in addition to the reticular fraction of STIM1 (84±2 kDa), the membrane and glycosylated form of STIM1 (90±2 kDa) for the SLE LBs (A) and for certain CLL diseases (B, mSTIM1+ group). This protein analysis first of all used an anti-STIM1 antibody, clone Gok/44 (BD Biosciences), then a peroxidase-coupled anti-mouse IgG antibody (GE Healthcare), and finally revealing by chimioluminescence (ECL advance kit, GE Healthcare).
C/D—The flow cytometry analysis consisted in incubating the purified LBs with an anti-STIM1 antibody, clone Gok/44 (BD Biosciences) for 15 min at 4° C., then, after washing, the binding of the anti-STIM1 antibody was revealed using a fluorescein-coupled anti-mouse IgG F(ab′)2 antibody (Jackson laboratories). The membrane labelling of STIM1 in the living cells is determined with respect to the isotype control (IgG2a, Beckman Coulter).