PBMCs were stained for surface markers and intranuclear IRF5. The cells were washed and stained with LIVE/DEAD fixable Near-IR dead cell stain (Invitrogen) and fluorochrome-conjugated surface antibodies (see Supplementary Table S2 ). Cells were fixed using 1% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. Fixed cells were first incubated with an anti-IRF5 antibody (E7F9W, Cell Signaling Technology) for 20 min and then a secondary anti-rabbit-IgG AlexaFluor488 antibody (Cell Signaling Technology) for 15 min. Stimulated samples and unstimulated controls were stained for intracellular cytokines with fluorochrome-labeled antibodies. Samples were acquired within 12 h on a BD LSRFortessa II (BD Biosciences). Cells were analyzed using FlowJo 10.7 software (BD Biosciences) with the exclusion of doublet cells. mDCs were identified as LD−, CD3−, CD14−, CD19−, CD20−, HLA-DR+, CD11c+, and CD123− cells. pDCs were identified as LD−, CD3−, CD14−, CD19−, CD20−, HLA-DR+, CD11c−, and CD123+ cells. Background staining was assessed for every sample by staining without the primary anti-IRF5 antibody (isotype control).
Anti rabbit igg alexafluor488 antibody
Manufactured by Cell Signaling Technology
The Anti-rabbit-IgG AlexaFluor488 antibody is a fluorescently labeled secondary antibody used in immunofluorescence applications. It is designed to specifically bind to rabbit primary antibodies, allowing for the detection and visualization of target proteins or cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Lsr fortessa 2, by BD (2 mentions)
Fixable near ir dead cell stain, by Thermo Fisher Scientific (1 mentions)
Anti igm bv421, by BioLegend (1 mentions)
Anti igd percp cy5, by BioLegend (1 mentions)
Anti cd3 buv737, by BD (1 mentions)
Anti cd20 pe cy7, by BioLegend (1 mentions)
Tcs sp5, by Leica (1 mentions)
Df12275, by Affinity Biosciences (1 mentions)
3 protocols using anti rabbit igg alexafluor488 antibody
1
Intranuclear IRF5 Expression in PBMCs
2
Flow Cytometric Analysis of IRF5 Expression in PBMCs
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PBMCs were stained for surface markers and intracellular IRF5. Cells were fixed using 1% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. Fixed cells were first incubated with anti-IRF5 antibody (Cell Signaling Technology, Cat#: 76983, clone E7F9W) for 20 min and then a secondary anti-rabbit-IgG AlexaFluor488 antibody (Cell Signaling Technology, Cat#: 4412) for 15 min. In the next step, the cells were washed and stained using fluorochrome-conjugated surface antibodies anti-CD3 BUV737 (BD Bioscience, Cat#: 564307, clone UCHT1), anti-IgM BV421 (Biolegend, Cat#: 314516, clone MHM-88), anti-IgD Per-CP-cy5.5 (Biolegend, Cat#: 348208, clone IA6-2), anti-CD20 PE-Cy7 (Biolegend, Cat#: 302312, clone 2H7) (see Additional file 1 : Table S1). Samples were acquired within 6 h on a BD LSRFortessa II (BD Biosciences). Cells were analyzed using FlowJo 10.7 software (BD Biosciences) with the exclusion of doublet cells. B cells were identified as CD3negative CD20 positive cells. The functionality of surface antibodies was compared on fixed and unfixed cells, showing a reliable result for all antibodies used (see Additional file 1 : Fig. S1).
3
Immunofluorescence Assay for GSDMD-N
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Caco‐2 cells were seeded on six‐well plates and exposed to experimental procedures. After washing, the cells were exposed to 4% paraformaldehyde for fixation as well as 0.2% Triton X‐100 for permeation. After that, cells were blocked with 5% bovine serum albumin solution for 1 h, and then cells were incubated with primary antibody against GSDMD‐N (DF12275, Affinity Biosciences) overnight at 4°C, following which was the probe with secondary anti‐rabbit IgG‐Alexa Fluor 488 antibody (#4412, Cell Signaling Technology) for 1 h at 37°C in the dark. Finally, DAPI solution was added to the cells away from light. Immunofluorescence photographs were acquired employing a confocal laser microscope (Leica, TCS SP5).
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