Anti cd16 32 antibody clone 93

Ear tissues were minced with scissors and digested with 2 mg/ml collagenase type 11, 500 µg/ml hyaluronidase and 100 µg/ml DNase (all from Sigma-Aldrich) at 37°C for 40 minutes. Cell suspensions were then filtered through a 200-mesh strainer and harvested for antibody staining. The single-cell suspension was firstly incubated with anti-CD16/32 antibody (clone 93, Biolegend) to block Fc-receptors and then stained with fluorescent antibodies for 30 min. The antibodies used were all purchased from Biolegend (San Diego, CA, USA) unless indicated: CD4 (clone GK1.5, Biolegend), Gr-1 (clone RB6-8C5), CD8 (clone 53-6.7), CD11b (clone M1/70) and TSLPR (clone 22H9). Dead cells were excluded by staining with 7-aminoactinomycin Viability Staining Solution (7AAD, Biolegend). The recovered number in one ear was calculated using Count Bright™ Absolute Counting Beads (Invitrogen, USA). Flow cytometry was performed on a BD FACS Calibur instrument (BD Biosciences, USA), and data was analyzed using FlowJo 7.6.4 software (Treestar).

Lung tissue leukocyte samples were subjected to viability staining and blocking of Fc receptors using the LIVE/DEAD fixable blue dead cell stain kit (Life Technologies) and an anti-CD16/32 antibody (clone 93; BioLegend). Cells were then washed and incubated with a staining mix containing antibodies against the following murine antigens: Siglec-F (phycoerythrin [PE], clone E50-2440; BD), Ly6G (Alexa Fluor 700, clone 1A8; BD), CD11c (allophycocyanin [APC], clone N 418; BioLegend), CD11b (BV421, clone M1/70; BD), CD4 (APC-Fire750, clone GK1.5; BioLegend), CD8a (CyChrome, clone 53-6.7; BD), CD3ε (biotin, clone 145-2C11; BioLegend), and NK1.1 (fluorescein isothiocyanate [FITC], clone PK136; BioLegend). Secondary staining was performed using streptavidin-BV605 and streptavidin-BV650 (BioLegend). All reagents and antibodies had been titrated before the experiments for optimal staining results. Flow cytometry data were acquired using LSRII and LSR Fortessa instruments (BD). Data were analyzed using FlowJo software (BD).

Neutrophil granulocytes were depleted by i.p. injection of 500 μg anti-Ly6G-antibody, clone 1A8 (BioXCell, #BE0075-1) and corresponding isotype control (InVivoMab Rat IgG2a, clone 2A3, BioXCell, #BE0089) 16 h prior to osmotic mini pump implantation.
For flow cytometric analysis of circulating neutrophils EDTA anticoagulated blood was collected 48 h after implantation of osmotic mini pumps and stained followed by lysis of red blood cells. Cells were fixed in 1% neutral buffered paraformaldehyde for 20 min at room temperature, according to a protocol by [6 (link)]. To prevent unspecific binding, cells were incubated with anti-CD16/32 antibody (clone 93; Biolegend, San Diego, CA, USA) before antibody staining.
CD11b-PE (M1/70, BD Biosciences, San Jose, CA, USA) and anti-Ly-6C-AlexaFluor^® 488 (HK1.4) were used to detect neutrophils and monocytes [21 (link)]. Respective isotype control IgGs were used to set all flow cytometric gates. Measurement and data analysis were performed using a Gallios™ Flow Cytometer, and Kaluza^® Flow Analysis Software (both Beckman Coulter Inc., Krefeld, Germany).

Ears of mice were separated in two sheets (ventral and dorsal) using forceps and enzymatically digested in RPMI 1640 medium containing 1 mg/ml collagenase (Sigma) and 50 ng/ml DNase (Sigma-Aldrich) for 60 min at 600 rpm and 37°C, and passed through a 70 μm cell strainer. Surface staining of cells was done by using APC or APC-Cy7 conjugated anti-CD45.2 (clone 104), APC, PerCP-Cy5.5 or APC-Cy7 conjugated anti-CD45.1 (clone A20), BV421 conjugated anti-F4/80 (clone BM8), Pe-Cy7, APC or APC-Fire conjugated anti-CD11c (clone N418), PE-Cy7 or APC-Cy7 conjugated anti-Ly6C (clone HK1.4), FITC conjugated anti-CCR2 (clone SA203G11), BV510 conjugated anti-MHC class II (IA/IE, clone M5/114.15.2), APC or APC-Cy7 conjugated anti-CD11b (clone M1/70), and PerCP-Cy5.5 conjugated anti CD45 (clone 30-F11), which were all purchased from BioLegend. Samples were Fc-blocked using anti-CD16/32 antibody (clone 93) (BioLegend) before antibody staining. Analysis was performed with a Fortessa or FACS ARIA III (BD Biosciences) using 405, 488, 561, and 633 nm lasers:. Photoconverted or non-photoconverted mKikume fluorescence was read out at 561 nm excitation and 610/20 nm emission, or 488 nm excitation and 530/30 nm emission, respectively. An autofluorescence signal was recorded at 488 excitation and 695/40 nm emission. Data were analyzed by using the FlowJo X software (FlowJo, LLC).

Single-cell suspensions of mouse lung tissue were prepared as previously described (Tsukui et al. 2020 (link)). Briefly, lung tissues were perfused with PBS through the right ventricle and then cut into pieces for digestion in Liberase TL (Sigma) and DNase I (Sigma) solution at 37 °C for 30 min. Then the cells were passed through a 70 μm cell strainer, washed, and suspended in precooled PBS with 1% fetal bovine serum. For lymphocyte isolation, density gradient centrifugation was performed using Percoll (Cytiva, USA). Before staining with antibodies, the cells were blocked with an anti-CD16/32 antibody (clone 93, BioLegend, USA). Viability was assessed using the Zombie NIR™ Fixable Viability kit (BioLegend, USA). All of the following fluorescence-conjugated antibodies were purchased from BioLegend and eBioscience (USA): anti-CD45(30-F11), anti-TCR-β (H57-597), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD69 (H1.2F3), anti-CXCR6 (3A051D1), anti-CD11b (M1/70), anti-Ly6G(1A8), anti-F4/80 (BM8), anti-iNOS (CXNFT), and anti-ARG1 (A1exF5). The iNKT cells were stained with fluorescence-conjugated mCD1d/PBS-57 tetramers provided by the National Institutes of Health (NIH) tetramer facility. Data were obtained from FACSVerse (BD Biosciences) and analyzed with FlowJo software (Tree Star).