Primary AML leukaemia cells were isolated from the bone marrow of AML patients at diagnosis from the Second Hospital of Anhui Medical University. Briefly, bone marrow blood was slowly layered over Ficoll‐Paque PLUS solution (GE Healthcare Life Sciences, Sweden) and centrifuged at 550×g for 25 mins. The mononuclear cells in the interphase layer were carefully transferred into another fresh tube, washed with Hank's balanced salt solution (Beyotime Biotechnology) twice and then applied to subsequent experiments. CD34+ haematopoietic stem/progenitor cells (HSPCs) and CD34− cells were purified from the cord blood of healthy donors from the First Affiliated Hospital of Anhui Medical University using Ficoll‐Paque PLUS and anti‐CD34‐coated magnetic beads (Miltenyi Biotec). Briefly, we isolated mononuclear cells from fresh cord blood as described above. Next, a single cell suspension obtained was incubated with anti‐CD34‐coated beads and selected on autoMACS Pro (Miltenyi Biotec). CD34+ cells were identified by incubation with CD34 antibody (BioLegend, USA) and analysis with flow cytometry (CytoFLEX, Beckman Coulter).
Anti cd34 coated magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD34-coated magnetic beads are a laboratory product designed to separate and isolate CD34-positive cells from biological samples. The beads are coated with antibodies specific to the CD34 antigen, which is expressed on the surface of hematopoietic stem and progenitor cells. These magnetic beads can be used in cell separation and purification procedures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Heat inactivated fbs, by Thermo Fisher Scientific (7 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (7 mentions)
Heat inactivated, by Thermo Fisher Scientific (7 mentions)
Tnf α, by R&D Systems (7 mentions)
Rpmi 1640, by Eurobio Scientific (7 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Hanks balanced salt solution (hbss), by Beyotime (1 mentions)
Ficoll paque plus solution, by GE Healthcare (1 mentions)
Ficoll paque plus, by Miltenyi Biotec (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Cd34 antibody, by BioLegend (1 mentions)
Facsaria, by BD (1 mentions)
Magnetic column, by Miltenyi Biotec (1 mentions)
Rpmi glutamax medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (1 mentions)
Flt3l, by Miltenyi Biotec (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
M csf, by Miltenyi Biotec (1 mentions)
10 protocols using anti cd34 coated magnetic beads
1
Isolation of AML and Healthy HSPCs
2
Mobilization and Isolation of CD34+ HSC
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Example 29
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
3
Mobilization and Expansion of CD34+ Stem Cells
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Example 38
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
4
Mobilization and Isolation of CD34+ HSC
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Example 29
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
5
Mobilization and Isolation of CD34+ Stem Cells
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Example 29
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+− enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
6
Mobilization and Expansion of CD34+ Stem Cells
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Example 38
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
7
Mobilization and Purification of CD34+ Stem Cells
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Example 29
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
8
Isolation and Culture of Dendritic Cell Subsets
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Blood CD34+ cells were isolated from PBMC by positive selection using anti-CD34-coated magnetic beads and magnetic columns according to manufacturer’s instructions (Miltenyi). CD34+ cells were cultured for 9–10 days in Yssel medium supplemented with 10% fetal calf serum (FCS), penicillin/streptomycin, 50 ng mL−1 GM-CSF (Miltenyi), 100 ng mL−1 Flt3-L (Miltenyi), and 10 ng mL−1 TNF-α (Miltenyi). DC subsets were isolated by cell sorting on a FACSAria instrument (BD Biosciences) after staining for CD1a and CD14. Monocytes (1×106 cells mL−1) were cultured for 5 days in RPMI-Glutamax medium (Gibco) supplemented with antibiotics (penicillin and streptomycin) and 10% FCS in the presence or absence of 100 ng mL−1 M-CSF (Miltenyi), 40 ng mL−1 IL-4 (Miltenyi), and 5 ng mL−1 TNF-α (Miltenyi). Cell population was isolated by cell sorting on a FACSAria instrument after staining for CD1a and CD16.
9
Mobilization and Purification of CD34+ Stem Cells
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Example 29
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
10
Isolation and Culture of CD34+ HSPCs
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CD34+ hematopoietic stem/progenitor cells (HSPCs) were sorted from the cord blood of healthy donors from the First Affiliated Hospital of Anhui Medical University using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Stockholm, Sweden) and anti-CD34-coated magnetic beads (Miltenyi Biotec, Bergishgradbach, Germany). Mononuclear cells were isolated from fresh cord blood as previously described. A single-cell suspension was incubated with anti-CD34-coated beads and selected on MACS (Miltenyi Biotec, Bergishgradbach, Germany). The purity of CD34+ cells was analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Miami, FL, USA) following incubation with anti-CD34 antibodies (BioLegend, San Diego, CA, USA). Primary CD34+ HSPCs were cultured in StemSpan SFEM medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 2 mmol/L L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% Lipid Mixture 1 (L0288, Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL SCF (Human origin, PeproTech, Rocky Hill, CT, USA), 2 ng/mL IL-3 (Human origin, PeproTech, Rocky Hill, CT, USA), and 1% penicillin-streptomycin.
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