6 well culture plate

Cells were seeded in a 6-well culture plate (Corning Costar Corp) to prepare for immunofluorescence (IF). After incubation with primary antibodies, the cells were incubated with fluorescence-labeled secondary antibodies. The slides were photographed using the inverted fluorescence microscope a TE-2000S (Nikon, Tokyo, Japan). DAPI and fluorescence-labeled secondary antibodies were obtained from Beyotime Biotechnology (Shanghai, China).

Stably transfected cells were seeded in the 6-well Culture plate (Corning Costar Corp, Corning, NY) to prepare for performed cell immunofluorescence (IF) and incubated with primary antibodies then incubated with fluorescence labeled secondary antibody. The slides were imaged using a microscope or inverted fluorescence microscope TE-2000S (Nikon, Tokyo, Japan). Primary antibodies for E-cadherin, vimentin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rhodamine-conjugated phalloidin, DAPI and fluorescence labeled secondary antibody were obtained from Beyotime Institute of Biotechnology (Shanghai, China).

For microscopy, 10⁶ THP-1 cells stably expressing YFP-ASC were plated in a 6-well culture plate (Costar) in a total volume of 2 ml/well. Cells were infected as described above, and the NLRP3 inflammasome activated by LPS + ATP treatment. ASC specks were visualized using the EVOS M7000 Imaging System, inverted microscope using 480/20 nm excitation and detection at 520 nm emission under a 40X objective. Fluorescent images of ASC specks were quantified using Image J software (NIH, http://rsbweb.nih.gov/ij).

Cellular immunofluorescence was performed as we described previously^{17 (link)}. Cells were seeded into the 6-well culture plate (Corning Costar Corp) to prepare for performing cell immunofluorescence (IF). After incubating with primary antibodies, cells then incubated with corresponding fluorescence labeled secondary antibody. After stained with DAPI (Beyotime Institue of Biotecchnology, Jiangsu, China), the slides were photographed using the inverted fluorescence microscope TE-2000S (Nikon, Tokyo, Japan). Primary antibodies for E-cadherin, vimentin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All fluorescence labeled secondary antibody were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Rhodamine-conjugated phalloidin (Beyotime Institute of Biotechnology) was used to analyze to cytoskeleton. Cells grown on cover slides were fixed, then incubated with Rhodamine-conjugated phalloidin (Beyotime Institue of Biotecchnology) and followed by DAPI (Beyotime Institue of Biotecchnology).

Life Technologies designed and synthesized the NFκB2 (p100) and the control siRNAs. The monocytes were transfected with siRNAs using the Amaxa Nucleofector system (Amaxa Biosystems). Briefly, 1 × 10⁶ monocytes, mixed with 25 μM of siRNA in 100 μL of transfection buffer, were transferred to an electroporation cuvette and nucleofected according to the manufacturer's instructions. The cells were then immediately transferred into a 6-well culture plate (Costar) containing 2 mL of prewarmed RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen). The nucleofected cells were cultured at 37°C with 5% CO₂ for 1 h before the assays.

Paired ovaries from euthanized PD2 or PD12 C57BL/6J female mice were dissected and washed three times in Leibovitz’s L-15 medium (Cat# 41400-045, Gibco) containing 10% fetal bovine serum (FBS) before being transferred individually to cell culture inserts (Cat# PICMORG50, Millipore) in a 6-well culture plate (Cat# 3506, Costar). One ovary was incubated with VCD and the contralateral ovary served as the paired control. Each cell culture insert housed three dissected ovaries. Ovaries were cultured in 1.5 ml Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) medium (Cat# 11039-021, Gibco) supplemented with 1 mg/ml BSA (Cat# A4161-1G, Sigma), 1 mg/ml AlbuMAX II Lipid-Rich BSA (Cat# 11021029, Gibco), 5% Insulin-Transferrin-Selenium (ITS-G) (Cat# 41400-045, Gibco), and 1% penicillin-streptomycin at 37°C with 5% CO₂ concentration and ∼95% relative humidity under normal atmosphere. The paraffin-embedded ovaries were serially sectioned into 5 μm thickness using a microtome and stained with hematoxylin for histological analysis.