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.