Perm2

Cells were washed and fixed using BD Cytofix buffer (cat. #554655), washed and permeabilized with BD Perm 2 (cat. #347692), washed and stained with PE-conjugated Ki67 antibody (BD Biosciences), and finally re-suspended in BD Cytofix buffer with Hoechst at 1 μg/mL. The cells were then analyzed on a BD LSRII machine with UV laser.

After stimulation, 2 mM EDTA (final concentration) was added to each well for 10 min at room temperature (RT). A LIVE/DEAD Fixable Dead Cell Stain (blue fluorescent reactive dye; Invitrogen) was used to identify and exclude non-viable cells from the analysis. Cells were fixed (BD Lyse Solution) and permeabilized (BD Perm 2), according to manufacturer's instructions. Surface and intracellular staining were done simultaneously for 30 min at RT with the following pre-titered antibody panel: anti–CD3 PerCP Cy5.5 (UCHT1; BD), anti–CD8 V450 (RPA-T8; BD), anti–CD4 V500 (RPA-T4; BD), anti–IFNγ PE-Cy7 (B27; BD), anti–IL-2 PE (5344.111; BD), anti–TNFα FITC (6401.1111;BD), and anti–MIP-1β APC (D21-1351; BD). Cells were acquired using an LSRII flow cytometer (BD) within 24 hours and ≥200,000 total events were collected for each sample unless otherwise specified.

Peripheral blood mononuclear cells were incubated with antibodies against CD3 (UCHT1) and CD28 (CD28.2) (all from Biolegend) to stimulate T cells, CD16 (3G8) (Biolegend) to stimulate NK cells, or F(ab′)2 anti-human IgM + IgG (eBioscience) to stimulate B cells in the presence of DMSO or inhibitors for 30 min on ice. Cells were washed and incubated with secondary Goat anti-mouse antibodies for 15 min, followed by stimulation for 2 min at 37°C. Cells were then fixed by BD Phosflow Fixation buffer and stained for extracellular markers with CD19-AF700 (HIB19), CD56-BV510 (NCAM16.2) and CD3-PerCP (UCHT1) (all from Biolegend). Cells were then permeabilized by BD Perm 2, stained with p-SLP-76 Y128 AF647 (J141-668.36.58) or p-SLP-65 Y84 AF647 (J117-1278) (both from BD) and analyzed by flow cytometry on a BD LSRFortessa.

As previously described (15 (link), 17 (link)), T cells with or without 8 h of restimulation were treated with monensin (2 µM) for the last 3 h, followed by staining with an appropriate combination of FITC-conjugated anti-KJ1-26, APC-conjugated anti-CD44, and PerCP-conjugated anti-CD4 mAbs. For staining, cells were washed once with FACS buffer (PBS with 3% fetal calf serum and 0.1% sodium azide) and then permeabilized with Perm2 (BD Biosciences) for 10 min at room temperature, followed by two washes in FACS buffer. Finally, cells were stained with an appropriate combination of anti-IFN-γ-APC and anti-IL-4-PE for 30 min at room temperature, washed, and resuspended in FACS buffer for analysis.

PBMCs were isolated from heparinized whole blood cells (HWB), carefully layered on Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and centrifuged for 30 min. Lymphocyte and monocyte layers were collected and stained using anti-CD3-FITC, anti-CD4-PE and anti-CD8-Pacific Blue antibodies (Biolegend, San Diego CA). Cells were permeabilized using Perm2 (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s recommendations and then stained for detection of intracellular IL-17A with an anti-IL-17A-PercP-Cy5.5 antibody (Biolegend). Samples were analyzed by flow cytometry.

At 6 hpi, cells were washed with PBS and harvested by trypsinization. After washing with fluorescence-activated cell sorter (FACS) buffer (PBS, 0.01% sodium azide and 0.1% bovine serum albumin (BSA), cells were fixed and permeabilized with Perm2 (BD Sciences, San Jose, CA, USA) for 10 min at room temperature (RT). Infected cells were detected after incubation with anti-p30 monoclonal antibody (diluted 1:100 in FACS buffer) for 30 min at 4 °C, followed by incubation with phycoerythrin (PE)-conjugated anti-mouse immunoglobulins (1:50, diluted in FACS buffer (Dako, Agilent Tech. Santa Clara, CA, USA) for 30 min at 4 °C. After extensive washing, 10,000 cells per tube in triplicates were scored and analyzed in a FACSCalibur flow cytometer (BD Sciences) to determine the percentage of infected cells under these conditions. The obtained infection rates were normalized to the corresponding control.

Vero cells were pretreated with inhibitors MG132, Lactacystin and Bortezomib at the indicated concentrations, followed by infection with Ba71V at a moi of 1 pfu/cell. Cells were washed with PBS after 90 min of adsorption at 37°C, and incubated with DMEM 2% 16 h. Cells were then harvested by trypsinization, and washed with flow cytometry buffer (PBS, 0.01% sodium azide, and 0.1% bovine serum albumin). Cells were fixed and permeabilized with Perm2 (BD Sciences) for 10 min at room temperature (RT). Detection of ASFV infected cells was performed by incubation with anti-p30 monoclonal antibody (diluted 1:100; kindly given by Dr. Escribano, INIA) or anti-p72 monoclonal antibody (1:1000; clone 1BC11 for immunofluorescence, Ingenasa) for 30 min at 4°C, followed by incubation for 30 min at 4°C with phycoerythrin (PE)-conjugated anti-mouse immunoglobulins (Dako) diluted 1:50. In order to determine the percentage of infected cells per condition, 10,000 cells/time point were scored and analysed in a FACS Canto II flow cytometer (BD Sciences). Infected cell percentages obtained after drug treatments were normalized to control values.