Anti mouse humancd45r b220

Splenic cells from C57BL/6
mice were isolated by forcing spleen tissue through the mesh into
PBS containing 2% fetal calf serum and 1 mM EDTA, and red blood cells
were depleted by lysis buffer. Cells were cultured in 96-well U-bottom
dishes (1 × 10⁶ cells/mL in RPMI 10% FCS) and incubated
with BTK inhibitors in different concentrations (1, 10, 100, 1000
nM) for 24 h at 37 °C in 5% humidified CO₂. Following
a 24 h incubation, cells were stimulated with anti-IgM overnight (5
μg/mL, Sigma-Aldrich). Subsequently, cells were stained with
anti-B220 (clone RA3-6B2, Biolegend) and anti-CD86 (clone GL-1, Biolegend)
antibodies (anti-mouse CD86 Biolegend 105008 1:400, anti-mouse/human
CD45R/B220 Biolegend 103212 1:400) for 30 min at 4 °C. Single-cell
suspensions were analyzed by a flow cytometer (CytoFlex, Beckman Coulter).

A full list of antibodies is provided in the Key Resource table (Table 1). The following antibodies were used for FACS analysis: Anti-mouse/human CD45R/B220, anti-CD23, anti-CD21/CD35 (CR2/CR1), anti-CD93 [AA4.1], anti-mouse IgM, anti-IgD, anti-CD45.1, anti-CD45.2, anti-CD3ε, anti-Ly-6G/Ly-6C(Gr-1), anti-CD11b, anti-TCRb, and anti-CD5 were all purchased from Biolegend. Anti-Mouse CD19 was purchased from BD Biosciences. Rabbit Anti-Ki67 (Novocastra), rabbit anti-cleaved caspase 3 (Cell signaling), rabbit anti-Phospho-Histone H3 (Ser10) (Cell signaling), rat anti-Pax5 (Biolegend), Guinea pig anti-Cytokeratin 8+18 antibody, and FITC rat Anti-mouse/human CD45R/B220 (BioLegend) were used for fluorescence immunohistochemistry. Anti-rabbit Alexa Fluor Cy3 (Jackson ImmunoResearch), anti-rabbit Alexa Fluor 647 (Jackson Immunoresearch), anti-rat Cy3 (Jackson Immunoresearch), and Alexa Fluor anti-guinea pig 647 secondary antibodies were used for in fluorescence immunohistochemistry. Notch2 (D76A6) XP (Cell signaling) and HRP-linked rabbit IgG (GE healthcare) secondary antibodies were used for western blot analysis.

Splenic cells from C57BL/6
mice were isolated by forcing spleen tissue through the mesh into
PBS containing 2% fetal calf serum and 1 mM EDTA, and red blood cells
were depleted by lysis buffer. Cells were cultured in 96-well U-bottom
dishes (1 × 10⁶ cells/mL in RPMI 10% FCS) and incubated
with ibrutinib, 1b, and 1f in different
concentrations (1, 10, 100, 1000 nM) for 24 h at 37 °C in 5%
humidified CO₂. Following a 24 h incubation, cells were
stimulated with anti-IgM overnight (5 μg/mL, Sigma-Aldrich).
Subsequently, cells were stained with anti-B220 (clone RA3-6B2, Biolegend)
and anti-CD86 (clone GL-1, Biolegend) antibodies (anti-mouse CD86
Biolegend 105008 1:400, anti-mouse/human CD45R/B220 Biolegend 103212
1:400) for 30 min at 4 °C. Single-cell suspensions were analyzed
by a flow cytometer (CytoFlex, Beckman Coulter).