Human cord blood (hCB) specimens were collected after elective caesarean delivery following full-term pregnancy at Beijing Union Hospital. All experimental procedures were approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-Sen University. Each participant signed the informed consent. CD38 and CD34 MicroBead kits (Miltenyi, Germany) were used to separate CD34 + CD38− cells from hCB and bone marrow.28 (link)
Cd34 microbead kit
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, United Kingdom, Canada
The CD34 MicroBead Kit is a laboratory product that enables the separation and enrichment of CD34-positive cells from a variety of sample types, including bone marrow, peripheral blood, and leukapheresis samples. The kit utilizes magnetic beads coated with antibodies specific to the CD34 cell surface antigen to facilitate the isolation of the target cell population.
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Lab products found in correlation
Ficoll paque plus, by GE Healthcare (13 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Macs cell separation system, by Miltenyi Biotec (8 mentions)
Ficoll paque, by GE Healthcare (7 mentions)
Ms column, by Miltenyi Biotec (7 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (7 mentions)
Bit 9500, by STEMCELL (6 mentions)
Ficoll paque plus, by Cytiva (6 mentions)
Lymphoprep, by STEMCELL (5 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Stemspan sfem, by STEMCELL (5 mentions)
Ls column, by Miltenyi Biotec (5 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (5 mentions)
Clinimacs, by Miltenyi Biotec (4 mentions)
Macs ld column, by Miltenyi Biotec (4 mentions)
Lymphoprep, by Axis-Shield (4 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (4 mentions)
Stemspan serum free expansion medium, by STEMCELL (4 mentions)
230 protocols using cd34 microbead kit
1
Isolation of Hematopoietic Stem Cells
2
CD34+ Cell Isolation from MNCs
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Human CB and mPB samples were obtained from healthy donors with informed consent. We got mononuclear cells (MNCs) using centrifugation on Lymphoprep medium. CD34 Microbead kits and LS columns using MACS magnet technology (Miltenyi) were used for MNC enrichment for CD34+ cell selection. Downstream experiments were conducted on CD34+ cells after sorting.
3
Hematopoietic Endothelial Differentiation from Pluripotent Stem Cells
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All experiments proceeded under authorization from the Institutional Review Board for Human Research at the CHA University (1044308‐202204‐LR‐023‐02) for CHA52 and Konkuk University (KUH1280081 for H1 and H9). Undifferentiated human PSCs (CHA52, H1, H9 embryonic stem cell line, PSCs) were seeded onto Matrigel®‐coated plates with mTeSR™1 medium (85850; StemCell Technologies Inc.) to differentiate HE from PSCs. The cells were incubated with APEL™ 2 medium supplemented with 3 nM − 1.5 μM CHIR‐99021 (S2924, Selleck Chemicals), 20 ng/ml of vascular endothelial growth factor (VEGF)165 (100‐20; PeproTech Inc.), and 25 ng/ml HumanKine® BMP‐4 (HZ‐1045; Proteintech Group Inc.) for 3 days to induce mesoderm.21 Based on previous, to induce and expand the HE, cells were then incubated with APEL™ 2 medium supplemented with 25 ng/ml HumanKine® BMP‐4 (HZ‐1045, Proteintech), and 250 ng/ml stem cell factor (SCF; 300‐07), 20 ng/ml VEGF165 (100‐20), 200 ng/ml FMS‐like tyrosine kinase 3 (Flt3)‐Ligand (300‐19), 100 ng/ml Thrombopoietin (TPO) (300‐18) and 20 ng/ml erythropoietin (EPO) (100‐64; all from PeproTech) as described previous papers.12 , 17 , 18 , 22 , 23 , 24 , 25 , 26 We isolated CD34+ cells from PSC‐derived HE by magnetic‐activated cell sorting (MACS) using CD34 MicroBead Kits (Miltenyi Biotec) as described by the manufacturer.27
4
Isolation and Expansion of hCBMCs
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Umbilical cord blood was obtained from normal vaginal and cesarean deliveries. Informed consent was received prior to the collection of cord blood and the study was approved by the Institutional Review Board of Chonbuk National University Hospital (Approval No. CBNUH: 2020-07-039-004). hCBMCs were generated as described previously 12 . Briefly, mononuclear cells were isolated by layering heparin-treated cord blood onto a Ficoll-Paque solution (GE healthcare, Chicago, IL, USA). CD34+ progenitor cells were isolated by magnetic cell sorting kit (CD34 Microbead kit, Miltenyi Biotech, Auburn, CA, USA). For the first week, CD34+ progenitor cells were cultured in Iscove modified Dulbecco medium supplemented with 1% insulin-transferrin-selenium, 0.1% β-mercaptoethanol, 50 ng/mL rhIL-3, 50 ng/mL rhIL-6, and 50 ng/mL rhSCF (all recombinant cytokines from PeproTech). After 6 weeks, the cells were cultured in 50 ng/mL rhIL-6 and 50 ng/mL rhSCF. hCBMCs cultured for at least 15 weeks were used for experiments, and cell purity was greater than 98%. hCBMCs were identified by flow cytometric analysis. Cell viability was determined by trypan blue (0.4%) exclusion.
5
Isolation of Human CD34+ Stem Cells
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Human cord blood was collected at the Mary Greeley Medical Center in Ames, Iowa, into 50 mL conical tubes containing 8 mL of anticoagulant citrate dextrose solution (38 mM citric acid, 85.25 mM sodium citrate, 136 mM dextrose). Mononuclear cells (MNCs) were isolated from cord blood by diluting blood 1:2 in HBSS and then layering over Ficoll-Paque (GE Healthcare). Buffy coats were collected and washed in HBSS. Prior to stem cell isolation, MNCs were resuspended in an isolation HBSS (iHBSS) consisting of 0.5% FBS and 2 mM EDTA in HBSS.
To isolate human CD34+ cells, we used a CD34 MicroBead kit from Miltenyi Biotech. Briefly, MNCs were incubated in iHBSS with CD34 microbeads and FcR blocking reagent for 30 min at 4°C on a rocker. After the incubation period, cells were washed in iHBSS and then passed through a LS column in a Miltenyi magnet (Miltenyi Biotech) to capture human CD34+ cells. The column was then removed from the magnet and cells were flushed, washed once more in HBSS, and then frozen in 10% DMSO and 90% FBS at −80°C until use.
To isolate human CD34+ cells, we used a CD34 MicroBead kit from Miltenyi Biotech. Briefly, MNCs were incubated in iHBSS with CD34 microbeads and FcR blocking reagent for 30 min at 4°C on a rocker. After the incubation period, cells were washed in iHBSS and then passed through a LS column in a Miltenyi magnet (Miltenyi Biotech) to capture human CD34+ cells. The column was then removed from the magnet and cells were flushed, washed once more in HBSS, and then frozen in 10% DMSO and 90% FBS at −80°C until use.
6
Isolation of CD34+ HSPCs from UCB
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Mononuclear cells were enriched from human UCB samples over Ficoll‐Hypaque gradient (GE Healthcare, UK) and CD34+ HSPCs were subsequently isolated using CD34 MicroBead Kit and MS Column (Miltenyi, Germany) according to the manufacturer's protocol. The purity was approximately 90% as evaluated by flow cytometry (BD Biosciences).
7
Isolation and Purification of CML-CP CD34+ Cells
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Proper informed consent was obtained from all participants. All experiments were performed according to the Declaration of Helsinki and were approved by the Ethics Committee of Juntendo University School of Medicine (IRB#2019113) and the Research Ethics Review Committee of the Institute of Medical Science of the University of Tokyo (2019-8-0702). Heparin-anticoagulated BM cells were obtained from 5 newly diagnosed CML-CP patients (Table S1 ). Informed consent was obtained in accordance with the Declaration of Helsinki, and all procedures were approved by the Research Ethics Board at Juntendo University. Mononuclear cells were isolated using Lymphoprep (STEMCELL Technologies) density gradient separation, and CD34+ cells (>85%) were enriched immunomagnetically using an CD34 MicroBead Kit (Milteny Biotec). Purity was verified by re-staining isolated cells with an allophycocyanin-cyanine7-labeled (APC-Cy7) anti-CD34 antibody (clone 561, BioLegend), and the cells were analyzed using a FACSAria (BD) machine.
8
Isolation and Culture of CD34+ Cells
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Peripheral blood samples were obtained from MF patients who provided consent to the use of their blood for research purposes. Patient clinical characteristics are summarised in Supplementary Table S1 . CD34+ cells were positively selected from mononuclear cells (MNC) using CD34+MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) as recommended by the manufacturer. CD34+ cells were cultured in StemSpan medium supplemented with CC-100 human cytokine cocktail (Stem Cell Technologies, Vancouver, Canada) for 48 hours (h) before use.
9
Isolation of Human BM-MNCs and AML Cells
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Normal human BM mononuclear cells (BM-MNCs) were obtained from allogeneic transplantation donors at the Institute of Hematology and Blood Diseases Hospital, CAMS/PUMC. Primary human BM AML cells were obtained from State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS/PUMC. Specimen acquisition was approved by Institutional Review Boards (IRB), State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS/PUMC. All donors signed the informed consent forms. The CD34+ cells were enriched using a CD34 Microbead Kit (Miltenyi, German).
10
Isolation of Human CD34+ CB Cells
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Human CD34+ CB cells were isolated using CD34 MicroBead Kit (Miltenyi Biotec, Cat: 130-046-703) according to the manufacturer’s instructions. Briefly, CB unit was diluted by sterile PBS to about 360 ml and aliquoted gently to human lymphocyte separation medium (DRKEWE, Cat: DKW-KLSH-0100) in 50 ml tube, ending with 35 ml CB dilution, and 15 ml lymphocyte separation liquid in each tube. The tubes were centrifuged at 1,500 rpm for 20 min. The monocytes were collected from the middle layer of the liquid and washed twice with PBS (CORNING, Cat: 21–040-CV) through centrifuging at 1,600rpm for 10 min. Then they were stained with equivalent CD34+ MicroBeads and FcR Blocking Reagent at 4 °C for 30 min. Incubated cells were washed with PBS for three times. Then, the cell suspension was loaded onto a MACS® Column, which was placed on the magnetic field of a MACS separator. After removing the column from the magnetic field, the magnetically retained CD34+ cells were eluted as the positively selected cell fraction into a new tube.
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