In vitro culture conditions maintaining the naïve B cell state. (A) Schematic diagram of the different steps of the in vivo CRISPR/Cas9 screening system. Briefly, naïve B cells isolated from donor mice of the indicated genotype are transduced with LV particles expressing sgRNAs specific for the genes of interest and a neutral control gene. After infection, the cells are cultured in vitro for 3 days. Transduced mCherry⁺ donor B cells are sorted by flow cytometry and transferred to recipient mice of the indicated genotype, which are immunized with the hapten NP to induce an immune response. The resulting mCherry⁺ plasmablasts are isolated, and the abundance of sgRNAs in plasmablasts is determined and compared with the abundance of the sgRNAs in the donor B cells. (B) Flow cytometric analysis of wild-type naïve splenic B cells cultured in vitro for 3 days under different conditions. B cells were cultured either in the B cell medium alone, or additionally in the presence of LPS, sBAFF, OP9 cells, or OP9 cells plus sBAFF. Before starting the culture, B cells were labeled with the CellTrace Violet dye. After 3 days, the cell viability, CD69 and IgD cell surface protein expression, differentiation into plasmablasts (CD138⁺CD22^–), and cell proliferation were assessed. (C) Summary of all the data generated with the different culture conditions indicated. The statistical data are shown as mean values with SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each dot corresponds to one mouse. Independent experiments were performed from two (IgD) to 10 (viability) times.