Isolation and Characterization of Lymphocytes from Bone Marrow and Spleen

Lymphocytes were isolated from cranial bone marrow using a previously reported protocol^{37 (link)}. In brief, the calvaria was cut into small pieces using sterile scissors and dissociated in PBS + 2% FBS with a pestle. Spleens were harvested from mice, washed in PBS and cut into 0.5 mm cubes in ice-cold PBS. Spleen or bone marrow isolates were transferred to a 70 μm cell strainer (Falcon, catalog no. 352350); cells were washed with PBS and resuspended in red blood cell lysis buffer (BioLegend, catalog no. 420301). Cells were stained for 30 min on ice using the following antibodies: anti-CD3 BV711 or anti-CD3-AF647 (clone okt3, BD Biosciences, catalog nos. 750983 and 566686)), anti-MuSK PE (clone 189-1 or 24C10), antihuman Ig light chain λ PE (clone MHL-38, BioLegend, catalog no. 316608), antihuman Ig light chain κ APC (clone MHK-49, BioLegend, catalog no. 316510), antihuman IgG PE (BD Biosciences, catalog no. 555787) and/or antimouse IgG APC (clone A85-1, BD Biosciences).