Anti cd19 v450

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.

About 50 mL of healthy human donor one (HD1) or healthy donor two (HD2) source leukocytes peripheral blood (Gulf Coast Regional Blood Center, Houston, TX, USA; E5318 product code) was used as a source for separating all nucleated cells from red blood cells using HetaSep (Stemcell Technologies) according to the manufacturer’s protocol. B cells were then extracted and enriched for from the nucleated cells using an EasySep Human CD19 + Positive Selection Kit (Stemcell Technologies) according to the manufacturer’s protocol. The enriched B cells were resuspended in ~500 µL of FACS buffer. These B cells were then stained with a 1:16 dilution of anti-IgD-PE (BD, cat#562024), a 1:16 dilution of anti-CD20-FITC (BD, cat#560962), a ~1:26 dilution of anti-CD19-v450 (BD, cat#560353), a 1:61 dilution of anti-CD3-PerCP-Cy5.5 (Biolegend, cat#300327), and a 1:16 dilution of anti-CD27⁻APC (BD, cat#558664) and then sorted for naive (CD20⁺CD19⁺IgD⁺CD3⁻CD27⁻) and memory (CD20⁺CD19⁺IgD⁻CD3⁻CD27⁺) cells on a FACSAria II.