Anti human cd33 pe

Prior to the analyses, erythrocytes were lysed with Red Blood Lysis Buffer (154 mM NH₄Cl, 14 mM NaHCO₃ and 0.1 mM EDTA). For flow cytometry analyses the following reagents were used: FcRBlocking Solution (BD Biosciences 553142); anti Human CD45 PerCp (BD Bioscience BMS45-9459-42); anti Human CD3 PE (BD Biosciences 555340); anti Human CD19 FITC (BD Biosciences 555412); anti Human CD33 PE (BD Biosciences 555450); anti Human CD11b FITC (BD Biosciences 562793) and, as an isotipic control, Simultest (BD Biosciences 342409). Flow-cytometric analysis was performed using the FACScan Beckton Dickinson and FlowJoX software. Blood cell counts were performed with the VetScanHM5 (ABAXIS). After 12 weeks, four mice of each cohort were sacrificed by cervical dislocation. Bone marrow from femur and tibia of these animals was sampled as described by Soleimani and Nadri [72 (link)] and collected in DMEM 10% FBS, 10 U/ml Heparin for flow-cytometry analysis and secondary transplant.
To monitor the secondary engrafment, flow-cytometric analyses of human CD45⁺ cells in the peripheral blood of the secondary recipients were performed 9 and 14 weeks after the transplant. On week 16, the mice were sacrificed and their bone marrow was collected and analysed.

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).