Tcs speii confocal module

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.