Anti ly6g percp cy5

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.

Lamina propria isolation from the small intestine of mice was performed by two treatments with HBSS + 2 mM EDTA, followed by treatment with collagenase VIII (0,6 mg/ml). The isolated cells (1 × 10⁶ cells per sample) were analyzed by flow cytometry for myeloid markers. The separation of alive/dead cells was possible by staining with the Fixable Viability Dye eFluor506 (eBioscience, Cat# 65-0866-14). The samples were further stained with the following antibodies: anti-CD45 AlexaFluor 700 (eBioscience, Cat# 56-0451-82), anti-SiglecF BUV395 (BD, Cat# 740280), anti-Ly6G PercpCy5.5 (BD, Cat# 560602), anti-Ly6C APC (eBioscience, Cat# 17-5932-80), anti-CD11b BV605 (BD, Cat# 563015), anti-CD64 BV711 (BioLegend, Cat# 139311), anti-F4/80 Biotin (eBioscience, Cat# 13-4801-82), Streptavidin BV421 (BioLegend, Cat# 405226), anti-CD11c PE-eFluor 610 (eBioscience, Cat# 61-0114-82) and anti-MHC ClassII APC-eFluor780 (eBioscience, Cat# 47-5321-80), lineage [CD19 (eBioscience, Cat# 15-0193-82), CD3 (eBioscience, Cat# 15-0031-82) and NK1.1 (BioLegend, Cat# 108716) PE-Cy5]. The cells were later fixed and processed for analysis using a five-laser BD LSRFortessa. The flow cytometry data were analyzed using FlowJo software 10.8.1 and presented as percentage to CD45+ population.