Visualizing NLRP3 and ASC in Activated Macrophages

Human monocyte-derived macrophages or differentiated THP-1 cells were cultured on coverslips and stimulated with PMA, LPS, ATP, or LPS plus ATP. Cells were fixed in 4% paraformaldehyde and processed for indirect immunofluorescence staining using the following Abs: mouse anti-human NLRP3 (1:100, ab16097, Abcam, Cambridge, MA, UK), mouse anti-human ASC (1:50, sc-514414, Santa Cruz Biotechnology), rabbit anti-human NCOA6 (1:500, A300-411A, Bethyl Laboratories, Montgomery, TX, USA), mouse anti-vimentin (1:100, ab8987, Abcam), and appropriate secondary Abs, such as donkey anti-mouse IgG Cy3 (1:200, 715-166-151, Jackson laboratory, Bar Harbor, MA, USA) and donkey anti-rabbit IgG Alexa 488 (1:1000, A21206, Invitrogen). The Golgi, ER, and lysosomes were stained using CellLight^TM Reagents BacMam 2.0 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The anti-NCOA6 Abs used in this study recognized the C-terminal end (amino acids 2000 to 2063) of human NCOA6 and thus are specific for only NCOA6α and NCOA6β out of four NCOA6 isoforms (α, β, γ, and δ), as these are the only isoforms that possess the recognized sequence [18 (link)]. Images were taken with a Zeiss LSM 810 microscope and contrast-enhanced using Zeiss ZEN microscope software.