Anti human cd15 pe

To validate the purity of neutrophils isolated from peripheral blood; 1x10⁷ CD15⁺ freshly isolated human neutrophils were resuspended in 100 ul FACS buffer (PBS, 0.5mM EDTA, 1% FBS, 0.1% BSA) and fresh whole human blood diluted 1:5 in FACS buffer and supplemented with 5 ul of human TruStain Fc receptor blocker (Biolegend, San Diego, CA) for 5 mins on ice. Cells were then incubated with anti-human CD15 PE (Biolegend) for 1 hour prior to FACS analysis. Cells were analyzed on the SORP FACS Symphony cell sorter (BD Biosciences) in the Flow Cytometry Facility at USC using FACS Diva software and all analyses was carried out in Flow Jo V10.8.0 (BD Biosciences).

HSPCs (CD34⁺) derived from individuals with LR-MDS and HR-MDS were transfected with 20 nM of SCR or mTOG-Ψ RNA oligos. The cells were harvested 6 h post transfection and 1 × 10⁵ cells were injected into sub-lethally irradiated (250 cGy) NSG-S mice (11–14 weeks old) via the tail vein. Human engraftment was followed in the peripheral blood of the transplanted mice every 2 weeks. After 8 weeks, the mice were killed and their BM cells were harvested, treated with ammonium chloride solution (StemCell Technologies) to lyse the red blood cells, and stained. The following antibodies were used: anti-mouse CD45–Alexa Fluor 700 (1:200; BioLegend), anti-human CD45–APC (1:200; BioLegend), anti-human CD19–BV605 (1:200; BD Biosciences), anti-human CD15–PE (1:200; BioLegend), anti-human CD33–PE (1:200; BD Biosciences), anti-human CD34–BV421 (1:100; BioLegend), anti-human CD123–BV605 (1:50; BioLegend) and anti-human CD45RA–FITC (1:200; Thermo Scientific). The cells were then washed, resuspended in 1 μg ml⁻¹ 7-AAD (BioLegend) in PBS + 3% FBS and analysed using an LSRFortessa X-20 flow cytometer (BD Biosciences).