Microscopic Detection of ASC Inflammasomes in Bone Marrow Derived Macrophages Post Stimulation

Abstract

An inflammasome is an intracellular multiprotein complex that plays important roles in host defense and inflammatory responses. Inflammasomes are typically composed of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), cytoplasmic sensor protein, and the effector protein pro-caspase-1. ASC assembly into a protein complex termed ASC speck is a readout for inflammasome activation. Here, we provide a step-by-step protocol for the detection of ASC speck by confocal microscopy in Bone marrow derived macrophages (BMBDs) triggered by chemical stimuli and bacterial pathogens. We also describe the detailed procedure for the generation of BMDMs, stimulating conditions for inflammasome activation, immunofluorescence cell staining of ASC protein, and microscopic examination. Thus far, this method is a simple and reliable manner to visualize and quantify the intracellular localization of ASC speck. Graphic abstract: Figure 1. Confocal microscopy detection of ASC speck formation in untreated WT BMDMs and WT BMDMs stimulated with LPS and ATP, transfected with dsDNA, and infected with F. novicida or Salmonella as indicated. Arrow indicates the ASC speck. Scale bars: 10 μm.

Keywords: AIM2; ASC; Confocal microscopy; Fluorescence staining; Inflammasome; NLRC4; NLRP3.

Figure 1.. Confocal microscopy detection of ASC speck formation in untreated WT BMDMs and WT BMDMs stimulated with LPS and ATP, transfected with dsDNA, and infected with F. novicida or Salmonella as indicated.

Arrow indicates the ASC speck. Scale bars: 10 μm.

Video 1.. Generation of BMDMs.

(This video was made at the Kunming Institute of Zoology according to guidelines from the Kunming Institute of Zoology on Animal Care and was approved by the Animal Research Ethics Board of the Kunming Institute of Zoology under protocol SMKX-20,6020.)