Anti cd16 cd32 antibody

For analyzing subset proportions, the monocytes treated with LPS for 5 days were harvested and incubated with anti-CD16/-CD32 antibodies (1:100 dilution, BD Biosciences, no. 553141) to block Fc-receptors. The cells were stained with fluorochrome-conjugated anti-CD11b (1:200 dilution, BioLegend, no. 101226) and anti-Ly6C (1:200 dilution, BioLegend, no. 128006) antibodies, and PI (Sigma-Aldrich) was added before flow cytometry. For examining S100A8 expression, the monocytes were treated with high-dose LPS (1 µg/mL), and IRF7 siRNA (Thermo Fisher Scientific), IRF3 siRNA (Thermo Fisher Scientific), or control siRNA (Thermo Fisher Scientific) was mixed with Lipofectamine reagent to transfect monocyte cultures (25 pmol siRNA/well). Fresh LPS and siRNA were added every two days. After 5 days, the cells were fixed, permeabilized, stained with fluorochrome-conjugated anti-S100Ab antibody (1:100 dilution, Novus, no. NBP2-27067AF647), and then analyzed by flow cytometry.

The following antibodies were used for flow cytometry analysis: CD45-AF700, CD25-PE, CD127-APC-eFluor780, CD3-SuperBright436, CD19-eFluor506, CD11b-PE/Cy5, Ly6C-AF488, Ly6G/Ly6C(Gr-1)-APC, Ly-6G-PE, Anti-Mouse/Rat Foxp3 FITC, and isotype control (Rat IgG2a K Isotype Control FITC) were procured from eBioscience (Waltham, MA USA); CD4-PE-Cy7 and CD8a-APC700 from BD Biosciences (San Jose, CA, USA).
Isolated cells from the spleens or tumors were incubated with anti-CD16/CD32 antibodies (BD Biosciences San Jose, CA, USA) to prevent non-specific antibody binding. Surface antigens were stained with the antibodies diluted in PBS containing 5% FCS and 2 mM EDTA and maintained at 4 °C. Intracellular stainings were performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Waltham, MA, USA).
Initially, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen). All samples were fresh samples, and all cells quantified were live cells (data not shown). We decided, subsequently, not to use LIVE/DEAD Fixable Aqua Dead Cell Stain to free one more channel for use.
Multiparametric assessment was conducted utilizing a Gallios flow cytometer (Beckman Coulter, Villepinte France), with subsequent analysis executed through the Kaluza® software. Flow cytometric evaluation of immune cells was restricted to live CD45+ cells following the exclusion of doublets.

After measuring PAP, mouse lungs were perfused by right ventricle administration of PBS, dissected, and digested as previously described [28 (link)]. Briefly, left lobes were digested with Liberase TM (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany), disrupted, and filtered using a 40 µm cell strainer (Corning^®, Somerville, NY, USA), followed by centrifugation for 10 min at 300× g. Red blood cells were lysed with ACK lysis buffer, and cells were resuspended in RPMI medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), filtered again, centrifuged, and resuspended in RPMI. Cell counting was performed using a Neubauer chamber after staining with trypan blue to exclude dead cells. For flow cytometry analysis, cells were resuspended in FACS buffer (PBS containing 5% bovine serum albumin and 0.1% sodium azide). Following the blocking of Fc receptors with anti-CD16/CD32 antibodies (BD Biosciences, San Jose, CA, USA), cells were stained with fluorochrome-conjugated antibodies to extracellular CD45, CD4, CD3, TCRβ, TCRδ, and intracellular staining for IL-6, IL-13, IL-17, and IFN-γ, all from BD Biosciences. Data were acquired with a BD FACSCalibur flow cytometer and analyzed using FlowJo™ v10 Software (BD Biosciences, FlowJo, LLC, Ashland, OR, USA) following the gating strategy as indicated in Supplementary Figure S2.

For staining, 2 × 10⁶ cells were stained with Zombie Aqua Viability Dye (BioLegend) in PBS for 10 min on ice, then Fc receptors were blocked with anti-CD16/CD32 antibodies from BD Biosciences (2.4G2) for an additional 10 min. After centrifugation, the supernatant was removed and cell were stained with a surface antibody cocktail containing in FACS buffer (PBS, 2 mM EDTA, 2% FBS) and Brilliant Stain Buffer Plus from BD Biosciences for 20 min on ice. The following antibodies were purchased from BioLegend; F4/80-PerCP/Cy5.5 (BM8), CD11c-PE/Cy7 (N418), CCR7-PE (4B12), CD90.2-A700 (30-H12), CD19-A700 (6D5), MHC-II-BV421 (M5/114.14.2), CD11b-BV605 (M1/70), CD8α-BV650 (53-6.7), Ly-6C-BV711 (HK1.4) and IL-12 PE (C15.6). CD40-FITC (HM40-3), CD103-APC (2E9), CD24-APC e780 (M1/69) and Granzyme B eFluor450 (NGZB) were obtained from Thermo Fisher Scientific. CD80-PE CF594 (16-10A1), CD45-BV786 (30-F11) and Ki-67 FITC (B56) were purchased from BD Biosciences. All samples were resuspended in FACS buffer and acquired on a BD Fortessa flow cytometer. Data were analyzed using FlowJo software from Tree Star, v10.5.

Cells were resuspended in 50 μl of staining buffer, and Fc receptors were blocked for 15 min at 4 °C with 10 μg/mL anti-CD16/CD32 antibodies (BD Biosciences.) After the blocking step, cells were labelled for 20 min at 4 °C with corresponding fluorochrome-conjugated monoclonal antibodies (Suppl. Table 1). The BD Pharmingen transcription factor buffer set was used according to the manufacturer’s protocol to detect expression of FoxP3. When necessary, cells were fixed in a final volume of 300 μl with 4% paraformaldehyde (PFA) for 10 min. For detection of intracellular cytokines, splenocytes were stimulated for 5 h with 500 ng/mL PMA (Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of 10 μg/ml brefeldin A (Biolegend). Then, cells were fixed and permeabilized with cyto Fix/Perm (Biolegend) and stained at RT for 30 min. Samples were acquired on a FACS Verse or a FACS Canto II cytometer (Beckton Dickinson), and data were analyzed with the FlowJo 7.6.5 software (TreeStar Inc.). Cell frequencies were determined by flow cytometry and absolute numbers calculated from counts determined using a hemocytometer (Malassez chamber).