Mononucleated cells were isolated from a pool of 10 fresh human cord blood (sex:F) using a Ficoll gradient. CD34+ cells were enriched using human CD34 MicroBeads (Miltenyi Biotec) and Auto MACS pro separator. CD34+ enriched cells were cultured in glucose-deprived IMDM + 4 mM glutamine + 10 mM [U-13C6] glucose or fructose, 10% dialyzed FBS, 1% Pen/strep and a cocktail of cytokines (100 nM SCF, 10 nM IL6, 10 nM FLT3-ligand, 10 nM GM-CSF, and 10 nM TPO) for 48 hours. The supernatant and the cell pellet were collected for further NMR and LC-MS analyses, respectively.
Human cd34 microbeads
Manufactured by Miltenyi Biotec
Sourced in United States
Human CD34 MicroBeads are a cell separation product developed by Miltenyi Biotec. They are designed for the isolation and enrichment of human CD34+ cells from various cell sources, such as peripheral blood, bone marrow, or cord blood, using magnetic separation techniques.
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Lab products found in correlation
Clinimacs, by Miltenyi Biotec (2 mentions)
13c5 citrulline, by Cambridge Isotopes (1 mentions)
Imatinib, by LC Laboratories (1 mentions)
13c6 arginine, by Cambridge Isotopes (1 mentions)
Bovine serum albumin (bsa), by STEMCELL (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
Thrombopoietin, by R&D Systems (1 mentions)
Flt3 ligand, by R&D Systems (1 mentions)
Interleukin 3 (il 3), by R&D Systems (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Cytotune 2, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Ficoll pacque, by GE Healthcare (1 mentions)
9 protocols using human cd34 microbeads
1
Metabolic Profiling of CD34+ Cells
2
CML Cell Culture Using Stable Isotopes
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Primary CML samples were thawed and recovered overnight using physiological medium (Plasmax) (Vande Voorde et al, 2019 (link)). This medium was supplemented with labelled or non‐labelled nutrients as well as standard supplements and growth factors as described previously (Kuntz et al, 2017 (link)), then filter sterilised through a 0.2 μM filter (Fisher Scientific: 10509821). Stable isotope tracers were purchased from Cambridge Isotopes (13C6 Arginine: Cat# CLM‐2265, 13C5 Ornithine: Cat# CLM‐4724 and 13C5 Citrulline: Cat# CLM‐8653) and added at concentrations found in Plasmax. Primary samples were seeded at a density of 400,000 cells/ml and cell lines at 100,000 cells/ml. For cell lines, the medium was supplemented with 10% dialysed FBS (Thermo Fisher Scientific Cat# A33820‐01). Imatinib was purchased from LC Laboratories and BCT‐100 was supplied from BCT International. The CD34+ cells were isolated using the CliniMACS (Miltenyi Biotec) to 95% purity while normal CD34+ cells (> 90% purity) using human CD34 MicroBeads (Miltenyi Biotec: Cat# 130‐100‐453), according to manufacturer's instructions.
3
Generation of hiPSCs from CD34+ Bone Marrow Cells
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To obtain hiPSCs, CD34+ cells were purified from primary human bone marrow (BM) cells (Allcells, Emeryville, CA, USA) via immunomagnetic separation (human CD34 microbeads; Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated CD34+ BM cells were transduced with OCT3/4, SOX2, KLF4, and c-Myc sendai virus (Cytotune 2.0; Thermo Fisher Scientific, Waltham, MA, USA) in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 15% bovine serum albumin, 1× BIT (Stemcell Technologies, Vancouver, Canada), 1% non-essential amino acids (Gibco), 100 ng/mL stem cell factor, 100 ng/mL thrombopoietin, 100 ng/mL Flt-3 ligand, and 20 ng/mL interleukin-3 (R&D Systems, Minneapolis, MN, USA) as previously described (19 (link), 20 (link)). After a 48 h incubation, transduced CD34+ BM cells were maintained on matrigel (BD Biosciences, Franklin Lakes, NJ, USA)-coated plates with TeSR-E8 iPSC culture medium (Stemcell Technologies). hiPSC colonies were manually selected ~15 days after transduction and expanded in TeSR-E8 medium.
4
Isolating CD34+ Cells from Embryoid Bodies
5
Isolation of CD34+ Cells from MNCs
6
Isolating CD34+ Cells from Embryoid Bodies
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Dissociated EB cells were thawed following the Lonza Poietics protocol and resuspended at 1×106 per 100 μL staining buffer (PBS + 2% FBS). CD34+ cells were sorted from bulk EB culture using human CD34 microbeads (Miltenyi Biotec) and run through a magnetic column separator (MACS) as per manufacturer’s instructions.
7
Targeting Leukemia Stem Cells in CML
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Leukemia cells from primary and relapse CML patients PB were incubated with human CD34 Microbeads (130-046-702, Miltenyi Biotec), and CD34+ CML cells (including LSCs and leukemia progenitor cells) were purified in sterile conditions. Indicated numbers cells were cultured in SFM containing SCF, TPO, Flt-3 ligand, and IL3 at 50 ng/mL and treated with various dose of free PM (2.5–20 nM) and BPTES (5 μM or 10 μM) for 48 h, then alive CD34+cells numbers were counted with trypan blue and FACS. Relative viable cells were normalized with that in control treatment group. For cell apoptosis assay, cells were stained with CD34 monoclonal antibody (Cat#12-0349-42, Biosciences) initially and then washed again with binding buffer before staining with Annexin-V and PI. The patient samples were obtained from West China Hospital which were approved by the clinical ethics committee in West China Hospital, Sichuan University. All patients signed the informed consent form, and met all requirements of the Declaration of Helsinki.
8
Isolation of CD34+ Cells from CML Patients
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Primary CML samples were sourced from CML patients either from 50 mL peripheral blood or leukapheresis product. Patients were in chronic phase CML at the time of diagnosis and gave informed consent in accordance with the Declaration of Helsinki and approval of the National Health Service (NHS) Greater Glasgow and CLyde Institutional Review Board. The CD34+ cells were isolated using the CliniMACS (Miltenyi Biotec) and purity verified to be >95% by flow cytometry (Apoptosis and CD34 analysis). Non-leukemic cells were isolated from femoral head material using human CD34 MicroBeads (Miltenyi Biotec), according to the manufacturer’s instructions. Purity was verified by flow cytometry to be >90%.
9
Isolation of CD34+ Hematopoietic Stem Cells
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CD34+ HSCs were isolated from human cord blood obtained from Department of Stem Cell Transplantation Research Lab, MD Anderson Cancer Center. Cell suspension from cord blood was incubated with human CD34 MicroBeads (Miltenyi Biotec). CD34+ HSCs were enriched via positive selection using Miltenyi Biotec MiniMACS Separator Columns (Miltenyi Biotec) according to the manufacturer protocol.
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