Cytofix cytoperm plus fixation permeabilization kit

Activated PBMCs were stained with anti-BDCA4-APC (CD304, Neuropilin-1, BioLegend, Cat. No. 354506) for 30 min at 4 °C then were fixed and permeabilized with BD Cytofix/CytopermTM Plus Fixation/Permeabilization Kit (BD Biosciences) according to the manufacturer’s instructions. After washing cells were incubated with anti-cleaved IL-1β (Asp116; clone D3A3Z; Cat. No. 83186S) or anti-cleaved caspase-1 (Asp297; clone D57A2; Cat. No. 4199S) antibodies (all from Cell Signaling) for 30 min at 4 °C. After washing cells were incubated with PE-conjugated IgG1 secondary antibody (Cat. No. 406421; BioLegend) for 30 min at 4 °C. As an isotype control rabbit IgG (Cell Signaling, clone DA1E, Cat. No. 3900S) was used. Stained cells were dissolved in Mowiol^® 4–88 mounting media (Sigma-Aldrich, Cat. No. 81381).
For confocal microscopy 100,000 cells were pipetted on 8-well μ-Slides (chambered coverslip, ibidi GmbH). Cells were visualized immediately by a Zeiss LSM880 confocal microscope. BDCA4-APC was excited at 633 nm and IgG1-PE was excited at 543 nm. Fluorescence emission was detected through 650 to 670 nm and 560 to 615 nm band-pass filters. Images were taken in multi-track mode to prevent cross-talk. Image stacks of 1024 × 1024 pixel, 1.5 μm thick optical sections were obtained with a 40× C-Apochromat water immersion objective (NA = 1.2).

DCs were seeded in an eight-well microtiter plate, cultured with or without Pmp18.1 for 24 h, harvested by digestion with accutase detachment solution (Thermo Fisher Scientific, Rockford, IL) and centrifuged at 1,500 g for 10 min. After washing thrice with FACS Buffer, cells were fixed and permeabilized using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit with BD GolgiStop™ according to the manufacture's protocol. The permeabilized cells were stained for 30 min on ice with mouse anti-NF-κB p65 monoclonal antibody (Santa Cruz, Dallas, Texas) washed with FACS Buffer, incubated with FITC-conjugated (green) goat anti-mouse IgG secondary antibody (BD Pharmingen, San Diego, CA) for 1 h on ice in the dark and counterstained with the nuclear stain, DAPI (Blue). The cells were washed with FACS Buffer, centrifuged to a slide, examined and photographed by immunofluorescence microscopy as previously described (Ekong et al., 2009 (link)).

TAMs from malignant ascites or pMPHs from peritoneal lavage fluid were stained with FITC-labeled anti-CD14 (Miltenyi Biotech), APC-labeled anti-CD206 (BioLegend), APC-labeled anti-HLA-DR or APC-labeled anti-CD206 (Biozol), PE-labeled anti-CD163, PE-labeled anti-CD64, PE-Cy7-labeled anti-CD16 and APC-labeled anti-CD32 (eBioscience) as described previously [4 (link)]. Intracellular staining was performed with PE-labeled anti-IL-10 (BD Biosciences) after permeabilization for 20 min at 4 C using BD Cytofix Cytoperm Plus Fixation Permeabilization Kit (BD Biosciences). Additionally, APC-labeled anti-CD52 or APC-labeled anti-TIMD4 (Biolegend) was used for surface staining of TAMs and MDMs from healthy donors. Isotype control antibodies were from BD Biosciences, Miltenyi Biotech and eBioscience. Cells were analyzed by flow cytometry and results were calculated as percentage of positive cells and mean fluorescence intensities (MFI).

Mouse lymphocytes from mesenteric lymph nodes and spleens were immunostained using fluorescently conjugated antibodies against CD4 (eBioscience, San Diego, CA, USA), IL-17 (eBioscience), interferon (IFN) γ (BioLegend, San Diego, CA, USA), and IL-4 (BD Biosciences, San Diego, CA, USA). Prior to intracellular staining, cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of Golgistop (BD Biosciences). Intracellular staining was performed using a BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and BD Golgistop Kit (BD Biosciences). The transcription factor Foxp3 was stained using a Foxp3/Transcription Factor Staining Kit (eBioscience) following the manufacturer’s protocol. Flow cytometry was performed with the aid of a cytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA).

To confirm the engraftment of human immune cells in mouse blood, flow cytometry was performed. To confirm the engraftment of human CD4 T cells, mononuclear cells in mouse blood were stained with human anti-CD4-PeCy7 (Cat. no. 300511; Biolegend, San Diego, CA). Four weeks after tissue transplantation, IL-17-expressing human CD4⁺ T cells (Th17) were analyzed in the whole blood of mice. Mononuclear cells in mouse blood were stained with human anti-CD4 and IL-17-PE (12-7179-42; eBioscience, San Diego, CA). Before intracellular staining, the cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiStop (554715; BD Biosciences, San Jose, CA). Intracellular staining was performed using a BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and BD GolgiStop Kit (554715; BD Biosciences). Flow cytometry was performed using a cytoFLEX flow cytometer (Beckman Coulter, Brea, CA), and the data was analyzed using FlowJo software (Tree Star, Ashland, OR).

Cell marker staining was performed using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences, Milan, Italy) according to the manufacturer's instructions. Briefly, 100 µL of cell suspension containing 5 × 10⁵ cells was used for each flow cytometric reaction. The antibody dilution, incubation and detection conditions are shown in Supporting Information 1 (Table 3). All samples were acquired with a FACS Calibur flow cytometer (Becton‐Dickinson, New Jersey, USA) and analysed with the CellQuest Pro software.