J774A.1 cells were stimulated as described above. After washing with PBS, the cells were fixed and permeated with 70–30 acetone–methanol as previously described (45 (link)). Briefly, cells were placed in a humidified slide chamber blocked for 1 h in 10% normal donkey serum (Jackson Immunologicals). After removal of the donkey serum, the primary antibodies to NLRP3 (Cryo-2; AdipoGen), caspase-1 (sc-514; Santa Cruz Biotechnology), and ASC (D2W8U; Cell Signaling) or isomolar, species-specific anti-IgG (R&D Systems, Inc.) were added and incubated overnight at 4 °C. The slides were then washed three times with PBS, and cells were incubated for 1 h at room temperature with donkey anti-mouse–Alexa555 (Life Technologies) or donkey anti-rabbit–Alexa488 (Life Technologies) conjugated secondary antibodies. Nuclei were stained with DAPI (Life Technologies). FRET images were acquired using a Marianas Imaging Station (Intelligent Imaging Innovations) using a Zeiss 639 Plan-Apochromat objective (1.4 N.A.), a Sutter Xenon light source, and a Cooke SensiCam (The Cooke Corporation) as previously described (45 (link)). The FRET was calculated as FRET = Transfer − (Fd/D) − (Fa/Aa) as reported (46 (link)).
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Immunofluorescence Imaging of NLRP3, Caspase-1, and ASC
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Corresponding Organization : Radboud University Nijmegen
Other organizations : University of Giessen, University of Colorado Anschutz Medical Campus, University of Colorado Health, Emory University, EIC Laboratories, J&S Studies, Olatec (United States), University of California, Los Angeles, Istituto Giannina Gaslini, Medizinische Hochschule Hannover, German Center for Lung Research
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Variable analysis
independent variables
- Stimulation of J774A.1 cells
- NLRP3 expression
- Caspase-1 expression
- ASC expression
- FRET (Förster Resonance Energy Transfer)
- Washing with PBS
- Cell fixation and permeabilization with 70–30 acetone–methanol
- Blocking with 10% normal donkey serum
- Incubation with primary antibodies (NLRP3, caspase-1, ASC, or isomolar, species-specific anti-IgG)
- Incubation with secondary antibodies (donkey anti-mouse–Alexa555, donkey anti-rabbit–Alexa488)
- Nuclei staining with DAPI
- Image acquisition using Marianas Imaging Station (Zeiss 639 Plan-Apochromat objective, Sutter Xenon light source, Cooke SensiCam)
- Isomolar, species-specific anti-IgG
- Isomolar, species-specific anti-IgG
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