ASC Speck Quantification and High Magnification Imaging

ASC Speck Quantification and High Magnification Imaging in Fixed Cells

1. Seed primary murine macrophages at a density of 1x10⁶ cells/cm² on 1.17 mm-thick glass bottom imaging plates in RPMI containing 10% FBS and 2% pen/strep (we seed 25,000 cells/well of a 384-well plate)
2. Allow cells to adhere for at least 4 hours or overnight
3. Stimulate cells as desired
   1. LPS/Nigericin: prime with LPS (100 ng/ml) for 2 hours prior to stimulation with Nigericin (1μM) for 1 hour
   2. LPS: Stimulate with LPS (10ng/ml) for 2 hours
   3. NOTE: For different stimulations, concentrations and timepoints will need to be optimized
4. Aspirate media and fix in 4% paraformaldehyde (PFA) for 15 minutes at room temp.
5. Aspirate PFA and block cells in blocking buffer (1X PBS, 5% FBS, 0.3% Triton X-100) for 1 hour at room temp.
6. Incubated overnight with anti-ASC antibody (Cell Signaling Technologies, 67824, 1:800 dilution in blocking buffer)
7. Wash plate 3x 5 minutes in PBS
8. Incubate 2 hours with Alexa Fluor 488-conjugated-Goat-anti-Rabbit IgG (Invitrogen A-11034, 1:1000 dilution in blocking buffer) and Hoechst 33342 fluorescent stain (1:10,000 dilution)
9. Wash plate 3x 5 minutes in PBS
10. Image cells on fluorescent microscope. NOTE: We use the BioTek Cytation3 and Lionheart automated microscope to image cells at 4X and 60X magnifications to allow for quantification and generate representative images respectively. Hoechst stain is detected at 350 nm and Hoechst+ nuclei are used to provide total cell count. The ASC signal is detected at 488 nm and ASC specks (1μm-3μm) are counted.

ASC Speck Quantification and High Magnification Imaging in ASC-GFP immortalized macrophages

1. Seed ASC-GFP immortalized macrophages at a density of 5x10⁵ cells/cm² on 1.17 mm-thick glass bottom imaging plates in RPMI containing 10% FBS and 2% pen/strep (we seed 12,500 cells/well of a 384-well plate)
2. Allow cells to adhere for at least 4 hours or overnight
3. Stimulate cells as desired in the presence of propidium iodide (1:100) and Hoechst stain (1:10,000)
4. Image cells on a temperature and CO₂ controlled fluorescent microscope such as the Cytation3 or Lionheart automated microscopes with built in environmental control to maintain 37°C, 5% CO₂ for the duration of the assay. Image cells at 4X and 60X magnifications at 15-minute intervals to allow for quantification and generate representative images respectively. Hoechst stain is detected at 350 nm and Hoechst+ nuclei are used to provide total cell count. The ASC signal is detected at 488 nm and ASC specks (1μm-3μm) are counted. Propidium iodide incorporation is detected at 617 nm and PI+ nuclei are counted to provide dead cell count.