Cell culture and treatment
Splenocytes were cultured in triplicate for 48 h in the presence or absence of the compounds and were stimulated with 5 μg/mL concanavalin A (Con A) to induce T cell proliferation and 10 μg/mL lipopolysaccharide (LPS) to induce B cell proliferation. The cells were pulsed with 0.5 μCi/well of [3H] thymidine for 8 h and harvested on glass fiber filters. The incorporated radioactivity was counted using a Beta Scintillation Counter (MicroBeta TriLux, PerkinElmer Life Sciences, Boston, MA, USA).
Bone marrow-derived macrophages (BMDMs) were differentiated as described previously. Briefly, BMDMs were separated from the femur and tibia bones of male C57BL/6J mice and then cultured for 7 days in RPMI-1640 medium containing 10% FBS and 10 ng/mL of M-CSF. Differentiation of THP-1 cells was induced by 100 ng/mL PMA for 48 h. The differentiated cells were treated with 1 μg/mL LPS in the absence or presence of PHI.
Immunofluorescence
For tissue samples, paraffin-embedded intestinal sections were dewaxed in xylene and rehydrated through gradient alcohols. After being blocked with 5% BSA, the tissue sections were stained with anti-F4/80. The signals were detected by goat anti-rabbit Alexa Fluor 647 conjugate and then counterstained with DAPI. For BMDMs, cells are fixed by 4% paraformaldehyde, permeated by 1% Triton X-100, and blocked by immunostaining blocking solution. Anti-NLRP3 is used to incubate cells at 4℃ overnight, followed by detection with goat anti-rabbit IgG Alexa Fluro 488 conjugate staining. The cell nucleus is then stained by DAPI. Captured images are analyzed using the Leica TCS SPS microscope and the Leica Application Suite X.