2.10. Cell apoptosis assay
SUDHL4 cells were treated with WK500B for 24 h. Cells were collected, washed with PBS resuspended in binding buffer andincubated with Annexin V-FITC and propidium iodide in the dark for 20 min. Samples were analysed immediately by using flow cytometry (FACS Calibur, BD Biosciences).
2.11. Immunization of mice
To induce the GC reaction, 8-week-old male C57BL/6 mice were immunized with 100 µg of 4-hydroxy-3-nitrophenylacetyl (NP)-(Santa Cruz) coupled chicken gamma globulin (CGG) (Cedarlane). Two days later mice were randomly assigned to the indicated groups and treated daily with drugs or vehicle (0.5% methyl cellulose) by intragastric gavage for 12 days. Mice were sacrificed at the peak of the GC response. Spleens were collected for flow cytometry and histology analysis, and serum was processed for ELISA assays.
2.12. GC analysis
Cell suspensions from spleens were prepared by grinding tissue through sterile wire mesh and stained with a panel of fluorescent-conjugated antibodies including PerCP/Cy5.5 conjugated anti-B220 (Biolegend, 103236), FITC-conjugated anti-FAS (BD Pharmingen, 554257), eFluor 660-conjugated anti-GL7 (Invitrogen, 50-5902-82), PerCP/Cy5.5 conjugated anti-CD4 (Biolegend, 100434 ), PE-conjugated anti-CXCR5 (Biolegend, 145504), and APC-conjugated anti-PD1 (Biolegend, 109112). Samples were run on a FACS Calibur (BD), and the data were analyzed with FlowJo software (TreeStar, Portland, OR).
2.13. Immunofluorescence histology
Mouse spleens were frozen in OCT compound (Sakura), and 6 μm-thick cryostat sections were stained with biotin-conjugated peanut agglutinin (Sigma) and IgD (BD Pharmingen) at 4 °C overnight followed by incubation with donkey anti-rat IgG(H+L)-Alexa Fluor 488 (Invitrogen) and streptavidin-Cy3 (Biolegend) for 2 h at room temperature. After washing in PBST, the slides were mounted in Prolong Gold anti-fade reagent (Molecular Probes). Images were acquired by a microscope, and ImageJ software (NIH) was used to quantify the GC areas.