Nigericin

BMDMs were obtained by culturing mouse bone marrow cells in culture media containing a 1:10 dilution of supernatant from the fibroblastic cell line CMG 14-12 as a source of macrophage colony-stimulating factor (86 ), a mitogenic factor for BMDMs, for 4-5 days in a 15-cm dish as previously described (85 (link)). Briefly, nonadherent cells were removed by vigorous washes with PBS, and adherent BMDMs were detached with trypsin-EDTA and cultured in culture media containing a 1:10 dilution of CMG for various experiments.
For all in vitro pharmacology experiments except otherwise specified, cells were pre-treated with vehicle (0.1% DMSO, final concentration) or inhibitors (in 0.1% DMSO, final concentration) for 1 hour before stimulation with the indicated ligand or ligands. Protein expression was analyzed by ELISA or Western blot as described below. To activate the NLRP3 inflammasome, BMDMs were plated at 10⁴ cells per well on a 96-well plate or 10⁶ cells per well on a 6-well plate overnight. Cells were primed with LPS and then with 15 μM nigericin (AdipoGen) as indicated, and conditioned media were collected for the analysis of IL-1β and LDH.

Asc-citrine, WT, and Nlrp3^CA/+;Gsdmd^−/− BMDMs were plated at 10⁴ cells per well on a 16-well glass plate overnight. Cells were primed with LPS for 3 hours and pretreated or not with CuET for 15 minutes before adding 15 μM nigericin (AdipoGen, CA) for 30 minutes. Cells were washed with PBS and fixed with 4% polyformalin buffer for 10 minutes at room temperature. For immunostaining, WT and Nlrp3^CA/+;Gsdmd^−/− BMDMs were permeabilized with 0.2% Triton in PBS for 20 minutes, blocked with 0.2% Triton, 1% BSA, and CD61 antibody (1:1000; Alexa Fluor 647, BD, NJ) in PBS for 30 minutes, and were incubated with ASC antibody (1:1000; clone 2EI-7, EMD Millipore, MA) overnight at 4°C in blocking buffer, followed by incubation with secondary antibody (Alexa Fluor 594, Life Technologies) for 30 minutes. Cells were counterstained with Fluoro-gel II containing DAPI (Fluoro-Gel, Fisher Scientific Intl INC, PA). Asc-citrine photographs were taken using ZEISS microscopy (Carl Zeiss Industrial Metrology, MN). ASC immunostaining images were taken using a Leica inverted microscope with a TCS SPEII confocal module and processed using LAS X software (Leica Microsystems Inc, IL). Quantification of ASC specks was carried out using ImageJ.