Anti human cd45 microbeads

Peripheral blood mononuclear cells (PBMC) were stained with ALDEFLUOR reagent (Stem Cell Technologies) for 30 min in a 37 °C water bath according to the manufacturer’s protocol. Cells were washed and subsequently stained for 30 min on ice with a mix of anti-human EpCAM-PerCP/cy5.5 antibody (Abcam, Cambridge, UK), anti-human CD45 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-human biotin-conjugated CD31 antibody (Invitrogen, Carlsbad, CA), anti-biotin microbeads (Miltenyi Biotec), anti-human CD45-APC antibody (BD Biosciences) and anti-APC streptavidin antibodies (BD Biosciences). Cells were washed twice with 1× aldefluor assay buffer. Leucocytes (CD45 + cells) and endothelial cells (CD31 + cells) were depleted using LD columns and MACS Separator (Miltenyi Biotec). BD LSR II (BD Biosciences) was used for cell analysis. Anti-human CD45-APC and anti-APC streptavidin antibodies were used as a precaution to detect and discard residual leucocytes or endothelial cells by FACs.

Glioblastoma tissues were obtained from the operating room and immediately dissociated using a papain-based dissociation system (Worthington Biochemical). Leukocytes were depleted using anti-human CD45 MicroBeads (Miltenyi Biotec). The C1 single-cell auto prep instrument (Fluidigm) was used for capturing single cells. Pre-amplified cDNA was utilized for qPCR with the Biomark HD system with IFC Controller HX (Fluidigm) and 2× SsoFAST EvaGreen supermix with low ROX (Bio-Rad).

To examine the effect of myelin-specific CD49d⁺CD154⁺ lymphocytes on OPC differentiation, 4 × 10⁴ lymphocytes were added to 2 × 10⁶ OPCs (1:50) which were seeded on six-well plates 24 h earlier. PMA (phorbol 12-myristate 13-acetate), an artificial stimulator of OPC differentiation, was added (0.1 mM) to culture in DMEM high glucose medium supplemented with 5% FBS, 1% penicillin–streptomycin at 37 °C with 5% CO₂ for 72 h. After incubation, supernatants were collected and cells were washed in DPBS. For further OL analysis, lymphocytes were removed by antihuman CD45 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Lymphocytes were attached to the magnetically polarized side of the Eppendorf tube and OLs remained at the tube’s bottom. These procedures allowed us to avoid OL activation during flowing through the magnetic column. OLs were transferred into a new tube, washed, and the dry pellet was frozen in −80 °C for NGS mRNA and miRNA ddPCR analysis.

Cells were incubated at 37°C in a humidified atmosphere with 95% air and 5% CO₂. OP9 stromal cells (CRL-2749) were cultured in α-MEM (HyClone) supplemented with 20% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 1× penicillin/streptomycin. Primary B-ALL cells harvested from xenografts were enriched via magnetic cell sorting using anti-human CD45 MicroBeads (Miltenyi Biotec) and cultured at a density of 1 × 10⁶/ml in RPMI1640 (HyClone, Thermo Scientific), 10% FBS, 2 mM L-glutamine, 1× P/S. For cytokine supplementation, the culture medium was supplemented with 10 ng/ml IGF-1 (Peprotech, 100–11), 10 ng/ml IL-7 (Peprotech, 200–07), 10 ng/ml IL-6 (Peprotech, 200–07). The culture medium was changed every two days. Adipocyte differentiation was induced following a previous study.^{21 (link)} Briefly, OP9 cells were cultured as a monolayer. Upon reaching confluence, the cells were maintained for an additional 2 days and then incubated in DMEM supplemented with 10% FBS, 1 µM Dexamethasone, .5 mM isobutyl-methylxanthine, 5 µg/mL insulin, and 1 µM Rosiglitazone (Sigma-Aldrich) for 2 days. Subsequently, the medium was replaced with DMEM plus 10% FBS and 1 µM Dexamethasone for 7–14 days, with fresh medium provided every 2 days.

Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with approved protocols from the institutional animal care and use committee of City of Hope National Medical Center. For the systemic AML model, 10⁶ luciferase-expressing Molm14 cells were injected into the tail vein of 6-to 8-week old NSG mice (Jackson Laboratory). To determine the leukemic burden, we injected the mice (I.P.) with 3 mg D-Luciferin (Promega) 4 days after tumor injection and imaged them in an IVIS 100 (Caliper Life Sciences). A standard region of interest (ROI), which included the entire mouse, was used to determine the total body bioluminescence. Data were expressed as photons/s/mm². The mice were then distributed into groups bearing equal tumor burdens and treated by gavage with NT1721 or the vehicle control (5% DMSO/30% solutol (Sigma)) as indicated.
To assess changes in gene expression levels in the human cells, we sacrificed mice after 14 days of treatment, extracted the bone marrow cells and isolated human cells using anti-human CD45 microbeads (Miltenyi) according to the manufacturer's protocol. The RNA was then isolated and used for qPCR assays.