Anti cd45 apccy7

Cells from lymph nodes (5x10⁵) or ear homogenates (1x10⁶) were washed with PBS at 400 g for 5 min at 4°C and blocked with Human FcX (BioLegend) for 15 min, followed by staining with the antibody cocktail for 30 min at 4°C. Cells were then washed with a cytometry buffer (PBS with 5% FBS) at 400 g for 5 min and 4°C), then fixed with 4% formaldehyde (Sigma) for 15 min at 4°C. Cells were washed and resuspended in the cytometry buffer and stored in the dark at 4°C until acquisition. The following antibodies were used: anti-PD-L1-APC, anti-CD10-APC-780 (human, eBioscience); anti-CD45-APCcy7, anti-CD11b-FITC, anti-CD11b-PE, anti-CD11b-PEcy7, anti-Ly6G-PerCP, anti-Ly6G-FITC and anti-PD-L1-APC (murine, eBioscience). Acquisition of events (lymph node, 100,000 events; ear, all cells) was performed on a BD FACSAria™. The gate strategy was performed based on the selection of cell size (FSC) and composition (SSC). After identifying the main population, a gate of FSC-A (area) and FSC-H (weight) was used, where cellular doublets were excluded. Gates for positive events were established through Fluorescence Minus One (FMO) control. The data analyzes were performed using the FlowJo software.

To assess the changes induced by prenatal betamethasone in leukocyte subsets of newborn mice, thymi and spleens of pups euthanised at d1 and d4 were harvested and weighed. Single-cell suspensions were obtained by mechanical disruption. Cells were stained for flow cytometry (FACS Canto II, BD Biosciences) analysis with anti-CD3e eFluor 450 (eBioscience, San Diego, CA, USA), anti-CD4 APC (BD Biosciences), anti-CD4 APC-Cy7 (eBioscience), anti-CD8α PE-Cy7 (eBioscience), anti-CD11b APC (eBioscience), anti-CD11c PE-Cy7 (BD Biosciences), anti-CD19 V450 (BD Biosciences), anti-CD25 PE (BioLegend, San Diego, CA, USA), anti-CD44 APC (eBioscience), anti-CD45 APC-Cy7 (eBioscience), anti-TCRγδ chain PerCP-Cy5.5 (BioLegend), anti-TCRαβ chain PerCP-Cy5.5 (BioLegend), anti-Ly6g PerCP-Cy5.5 (BD Biosciences) and Pacific Orange dye (ThermoFisher Scientific, Waltham, MA, USA) for viability. Data were manually analysed using FlowJo software (Tree Star Inc., Ashland, OR, USA). The dimensionality reduction algorithm t-SNE (t-distributed stochastic neighbour embedding) in R, using the CRAN package “Rtsne”, was used for direct comparison of treated and untreated samples.

10 ml of PBS was perfused through the left ventricle of mice while venous runoff was collected from the orbit. Nasal bones were dissected and placed in 1 ml of DMEM (5% FBS + 1 mg/ml collagenase D (Roche)). Samples were incubated for 45 minutes at 37°C and disaggregated suspension over a 70 µm mesh screen. 2×10⁶ cells per well were added to 96 well plates. Samples were resuspended in 200∶1 anti-CD16/32 (BD Biosciences) in PBS + 2% FBS and incubated on ice for 20 minutes. Following wash, cells were incubated with the following antibodies in PBS + 2% FBS: (All antibodies purchased from BD Bioscience unless otherwise stated) anti-CD45:APC-cy7, anti-CD11b:Horizon V450, anti-Ly6G: APC (E Bioscience), anti-CD11c:Per-CP, Anti-F480:PE-Cy7 (E Bioscience) Anti I-A^d:PE (E Bioscience), Anti-CD3:Horizon V450, anti-NK1.1:AF-700.