Conditional YY1 Knockout in Murine Splenic B Cells

Follicular B cells were purified from mouse spleen with anti-CD23-biotin (Biolegend) and streptavidin microbeads (MACS; Miltenyi Biotec). Conditional YY1 knockout in splenic B cells was performed ex vivo using TAT-CRE enzyme purified from bacteria, as previously described [33 (link), 34 (link)]. Briefly, cells were washed three times with opti-MEM (Invitrogen) and then incubated with TAT-CRE recombinant protein for 45 min at 37°C. To inactivate TAT-CRE, fetal bovine serum was added to a final concentration of 10%. Cells were washed with splenic B medium (RPMI 1640, 10% HyClone fetal bovine serum (Thermo Scientific), sodium pyruvate, 55 μM 2-mercaptoethanol (Sigma), minimal essential medium (MEM) nonessential amino acids, 2 mM l-glutamine, 1% Penicillin-Streptomycin (Invitrogen), and then cultured at 37°C in a 5% CO₂ atmosphere. Cells were activated ex vivo with 10 μg/mL bacterial LPS (Sigma) plus 20 ng/mL IL4 to stimulate proliferation and CSR.