Anti cd86 clone gl 1

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).

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, IbrCl-1342 in different concentrations (1, 10, 100,
and 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)
and 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).

Antibodies were all purchased from BD Biosciences, unless indicated otherwise. Briefly, cells were first incubated with anti-mouse CD16/32 (Fc block) for 15 min at 4°C and then stained with FITC, PE, APC or PerCP.Cy5.5 conjugates of anti-MHC-II (clone 2G9), anti-CD11c (clone HL3, Biolegend), anti-F4/80 (clone CI:A3-1), anti-CD80 (clone 16-10A1), anti-CD86 (clone GL1) or anti-CD11b (clone M1/70) for 30 min at 4°C. For unconjugated anti-CD120a (TNFRp55, clone 55R-286) or anti-CD120b (TNFRp75, clone TR75-89) antibodies, the cells were stained a second time with PE-conjugated anti-hamster polyclonal antibody.
For intracellular staining of splenocytes, the cells were stimulated with PMA/ionomycin (50/750 ng/ml, eBioscience) for 5 h in the presence of GolgiPlug (BD Biosciences). After washing, cells were stained with anti-MHC-II and anti-CD11c, fixed with 1% paraformaldehyde, and then a permeabilizing solution BD FACS (BD Biosciences) was applied for intracellular staining with anti-IL-12/23p40 (clone C15.6).
For detection of Th1 and Th17 lymphocytes, the cells were stained with anti-CD4 (clone RM4-5) and anti-CD3 (clone 145-2C11), fixed and permeabilized as described above using anti-IL-17A (clone eBio17B7C15.6, eBioscience) or anti-IFN-γ (clone XMG1.2).
Data were obtained on a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).