Anti cd33 apc

For the in culture differentiation assay, cells were transfected with mTOG or SCR oligos on days 0 and 7 of the protocol. The differentiation assay was performed as previously described^{34 (link)}. Briefly, the cells were expanded for 7 d in stem cell medium (IMDM (Sigma), 15% BIT (StemCell Technologies), 1% GlutaMAX (Invitrogen), 10 ng ml⁻¹ IL-3 (Peprotech), 10 ng ml⁻¹ IL-6 (Peprotech) and 25 ng ml⁻¹ SCF (Peprotech); the cells were maintained at a concentration of 0.1 × 10⁶ cells ml⁻¹. After 7 d, the cultures were divided for erythroid and myeloid differentiation. Erythroid differentiation of cells at a concentration of 0.2 × 10⁶ cells ml⁻¹ was induced using stem cell medium supplemented with 2 U ml⁻¹ erythropoietin (Peprotech). Myeloid differentiation of cells at 0.3 × 10⁶ cells ml⁻¹ was induced in Myelocult H5100 medium (StemCell Technologies) supplemented with 1 × 10⁻⁶ M hydrocortison (StemCell Technologies) and 20 ng ml⁻¹ G-CSF (Peprotech). Differentiation was evaluated in healthy control BM samples by May–Grünwald Giemsa staining and flow cytometry as a positive readout. For erythroid and myeloid assessment, the following antibodies were used: anti-CD36–PE (1:200; BioLegend), anti-CD235a–PECy7 (1:1,000; BioLegend), anti-CD33–APC (1:200; BioLegend), anti-CD66b–FITC (1:200; BD Biosciences) and anti-CD34–BV421 (1:100; BioLegend).

Red blood cells were lysed using ammonium chloride solution according to manufacturer’s instructions (Qiagen, Cat# 1,045,722) for 10 min at room temperature prior to antibody staining. Tumour and tissue samples were digested using Type II collagenase (Sigma; Cat#C6885-100MG;) for three hours at 37 °C, washed and re-suspended in RPMI + 10% FBS; R10% (RPMI-1640 (Sigma-Aldrich; Cat# R8758) with 10% heat-inactivated fetal bovine serum, glutamine (1X), sodium pyruvate (1X) and Penicillin–Streptomycin (RPMI 10% = R10%). Immune populations were identified by staining with anti-human antibodies: anti-CD33-APC (BioLegend Cat# 303,408, RRID:AB_314352), anti-HLA-DR-PE (BD Biosciences Cat# 555,812, RRID:AB_396146), anti-CD14-PE-Cyanine7 (Thermo Fisher Scientific Cat# 25-0149-42, RRID:AB_1582276), anti-CD68-Alexa Fluor 488 (BioLegend Cat# 333,812, RRID:AB_2074832), anti-CD163-Brilliant Violet 421 (BioLegend Cat# 333,612, RRID:AB_2562463), anti-CD206-APC/Cyanine7 (BioLegend Cat# 321,120, RRID:AB_2144930) and anti-CD15-FITC (BioLegend Cat# 301,904, RRID:AB_314196) antibodies on ice for 30 min in Flow Buffer consisting of 1X PBS (Sigma-Aldrich: P3813) and 2%BSA (Sigma- Aldrich: 9048-46-8). Cells were acquired using a Cyan and CytoFLEX flow cytometers and analysed using FlowJo and CytoExpert2.2.