Anti nlrp3 primary antibody

Elicited murine peritoneal macrophages exposed to crocidolite asbestos fibers at a concentration of 20 μg/cm² were assessed for induction of the NLRP3 inflammasome 24 hours following asbestos exposure. This was done by fixing the cells (1 : 1 methanol-acetone fixation) and immunostaining for the NLRP3 subunit by using polyclonal anti-NLRP3 primary antibody (catalogue number 15101S, Cell Signaling Technology, Danvers, MA, USA) and the goat anti-rabbit-Alexa 488 secondary antibody (Abcam, Cambridge, MA, USA) followed by imaging on a Zeiss LSM510 scanning laser microscope. All images were acquired at the same exposure and offset settings using LSM Metamorph Imaging® software (Molecular Devices, Sunnyvale, CA, USA). The fluorescent images of cells were processed and quantitated for NLRP3 expression by the use of ImageJ software (NIH). The intensity of cells in each field was integrated to obtain the total fluorescence intensity of a particular field. Three to four fields were imaged for each condition (control, LGM2605 only, asbestos treated, asbestos, and LGM2605) for n = 3 independent experiments.

Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Thermo Scientific (Waltham, MA, USA). Modified radioimmunoprecipitation assay (RIPA) lysis buffer, protease inhibitor cocktail, Coomassie Plus™ Protein Assay Reagent, and chemiluminescent immunoblotting reagent were obtained from Thermo Fisher Scientific (Rockford, IL, USA). QIAzol lysis reagent was purchased from Qiagen (Valencia, CA, USA). ReverTra Ace^® qPCR Master Mix was purchased from Toyobo Co., Ltd. (Osaka, Japan). SensiFAST^TM SYBR^® Lo-ROX Kit was purchased from Meridian Bioscience^® (Cincinnati, OH, USA). The MTT or 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye and mouse anti-β-actin primary antibody were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Anti-NLRP3 primary antibody, anti-ASC primary antibody, anti-caspase-1 primary antibody, anti-NF-κB primary antibody, anti-PARP primary antibody, and goat horseradish peroxidase-conjugated anti-mouse- or anti-rabbit-IgG were obtained from Cell Signaling Technology company (Danvers, MA, USA).