Pe anti human asc clone hasc 71

PBMCs (1 × 10⁶), stimulated as described above, were fixed with 100 μL of Paraformaldehyde (PFA) (1%) (BDH, United Kingdom), permeabilized with 100 μL of saponin (0.1%) (Life Science VWR, Lutterworth, Leicestershire, LE), and stained with PE-antihuman ASC (clone HASC-71, isotype mouse IgG1, Biolegend, San Diego, CA, United States) for 1 h at room temperature; cells were then washed with PBS, centrifuged at 1,500 rpm for 10 min, resuspended in 50 μL of PBS, and examined using the AMNIS FlowSight Imaging Flow Cytometer (Luminex Corporation Austin, TX). Results were analyzed using an analysis software (IDEAS). The IDEAS image analysis software allows quantification of cellular morphology and fluorescence at different cellular localizations by defining specific cellular regions (masks) and mathematical expressions that uses image pixel data or masks (features).

PBMCs (1 × 10⁶), stimulated as described above, were fixed with 100 µl of PFA (1%) (BDH, UK), permeabilized with 100 µl of Saponine (0.1%) (Life Science VWR, Lutterworth, Leicestershire, LE), and stained with FITC-anti human NLRP3 (Clone #768319, isotype Rat IgG2a, R&D Systems, Minneapolis, MN, USA) and PE-anti human ASC (clone HASC-71, isotype mouse IgG1, Biolegend, San Diego, CA, USA) for 1 h at room temperature; cells were then washed with PBS, centrifuged at 1,500 rpm for 10 min, resuspended in 50 µl of PBS, and examined using the AMNIS Flowsight Imaging. Results were analyzed by IDEAS analysis software (Amnis Corporation, Seattle, WA, USA).
The analysis of NLRP3 expression was performed by internalization feature utilizing a mask representing the whole cell, defined by the brightfield image, and an internal mask defined by eroding the whole cell mask.
Apoptosis-associated speck-like protein containing CARD speck formation was analyzed using the same mask of internalization feature, differentiating diffuse or spot (speck) fluorescence inside of cells. Threshold mask was used to separate all ASC positive cells population in ASC-Speck spot cells or ASC-diffuse cells by the different diameter of the spot area: in ASC-speck, the spot shows a small area and high max pixel vice versa in cell with ASC-diffuse.

PBMC (2 × 10⁶), stimulated as described above, were fixed with 100 μL of PFA (1%) (BDH, UK), permeabilized with 100 μL of Saponine (0.1%) (Life Science VWR, Lutterworth, Leicestershire, UK), and stained with FITC-anti human NLRP3 (Clone #768319, isotype Rat IgG2a, R&D Systems, Minneapolis, MN, USA) and PE-anti human ASC (clone HASC-71, isotype mouse IgG1, Biolegend, San Diego, CA, USA) for 1 h at room temperature; cells were then washed with PBS, centrifuged at 1500 rpm for 10 min, resuspended in 50 μL of PBS, and examined using the AMNIS FlowSight. Results were analyzed by IDEAS analysis software (Amnis Corporation, Seattle, WA, USA). The analysis of NLRP3 expression was performed by internalization feature, utilizing a mask that represents the whole cell defined by the brightfield (BF) image and an internal mask defined by eroding the whole cell mask. Apoptosis-associated speck-like-protein containing CARD speck formation was analyzed using the same mask of internalization feature, differentiating diffuse or spot (speck) fluorescence inside of cells. Threshold mask was used to separate all ASC positive cells population in ASC-speck spot cells or ASC-diffuse cells by the different diameter of the spot area: in ASC-speck, the spot shows a small area and high max pixel, and the opposite occurs in cells with ASC-diffuse.