The human leukemic cell lines K562 and HL-60 were obtained from the Korean Cell Line Bank (Seoul National University, Seoul, Korea), and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Human hematopoietic progenitor CD34+ cells were purchased from STEM CELL Technologies (Vancouver, BC), and cultured in Hematopoietic Progenitor Expansion Medium DXF with cytokine mix E (PromoCell, Heidelberg, Germany).
Cd34 cells
Manufactured by STEMCELL
Sourced in Canada
CD34+ cells are a specific population of human hematopoietic stem and progenitor cells. They express the CD34 surface antigen and are involved in the formation of various blood cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cytokine mix e, by PromoCell (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Hl 60, by Korean Cell Line Bank (1 mentions)
Gentamicin, by Euroclone (1 mentions)
Glutamine, by Euroclone (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Ficoll, by Merck Group (1 mentions)
Rpmi 1640, by Euroclone (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (1 mentions)
Stempro 34 sfm, by Thermo Fisher Scientific (1 mentions)
Methocult classic, by STEMCELL (1 mentions)
Pacific blue anti human cd3, by BioLegend (1 mentions)
Fitc anti mouse cd45, by BioLegend (1 mentions)
7 aad, by BioLegend (1 mentions)
Apc anti human cd45, by BioLegend (1 mentions)
Pe anti human cd13, by BioLegend (1 mentions)
Apc cy7 anti human cd19, by BioLegend (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
16 protocols using cd34 cells
1
Culturing Leukemic and Hematopoietic Cells
2
Isolation of Mononuclear Cells from Blood
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Normal human umbilical cord blood (CB) samples were obtained from the Royal London Hospital under ethical approval (HRECO6/Q0604/110) and from the Anthony Nolan Research Biobank (15/EM/0045). Thymus tissue was obtained from the Evelina Children’s Hospital (09/H0504/39). Blood from children was obtained in collaboration with Dr. C. Furness and Prof. M. Greaves at the Institute for Cancer Research using United Kingdom Childhood Cancer Study stored samples from patients with nephroblastoma or neuroblastoma (CCR2285); adult blood was obtained from healthy volunteers (07/H0803/237). T cell acute lymphocytic leukemia (T-ALL) samples were obtained from the Bloodwise Childhood Leukaemia Cell Bank (16/SW/0219). Mononuclear cells were isolated from blood by centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences). For CB, mononuclear cells were enriched in CD34+ cells (STEMCELL Technologies), and the CD34− fraction (on occasion from pooled donors) was used for experiments.
3
Isolating Leukemic Blasts for Research
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Leukemic blasts cells were obtained from the peripheral blood or bone marrow of leukemia patients and purified by Ficoll (Sigma-Aldrich). Cells were growth in RPMI 1640 (Euroclone) supplemented with 20% heat-inactivated FBS (Sigma-Aldrich), 1% glutamine (Euroclone), 1% penicillin/streptomycin (Euroclone), and 0.1% gentamicin (Euroclone). The experiments were approved by the University of Campania “Luigi Vanvitelli” Ethical Committee (Prot. number: 296). CD34+ cells were purchased from STEMCELL Technologies (#70002).
4
Culturing Human Mast Cells from CD34+ Cells
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LAD2 MC [22] were cultured in serum free media (StemPro-34 SFM, Life Technologies) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 µg/ml streptomycin and 100 ng/ml stem cell factor (SCF). The cell suspensions were seeded at a density of 105 cells/ml and maintained at 37°C and 5% CO2. Cells were fed by hemi-depletion of medium once per week.
Human peripheral blood derived CD34+ cells (StemCell Technologies, Vancouver, Canada) were cultured in StemPro-34 SFM supplemented with 2 mM L-glutamine, 50 µg/ml streptomycin, 100 U/ml penicillin, 100 ng/ml SCF, and 100 ng/ml recombinant human IL-6 (PeproTech, Inc., Rocky Hill, NJ). Recombinant human IL-3 (30 ng/ml) was added for the first week. Half of the culture medium was replaced every 7 days. Cultures at 8 to 10 weeks consisted of greater than 99% huMC [23] (link). Unless otherwise stated, experiments were performed in StemPro-34 SFM complete with 100 ng/mL SCF.
Human peripheral blood derived CD34+ cells (StemCell Technologies, Vancouver, Canada) were cultured in StemPro-34 SFM supplemented with 2 mM L-glutamine, 50 µg/ml streptomycin, 100 U/ml penicillin, 100 ng/ml SCF, and 100 ng/ml recombinant human IL-6 (PeproTech, Inc., Rocky Hill, NJ). Recombinant human IL-3 (30 ng/ml) was added for the first week. Half of the culture medium was replaced every 7 days. Cultures at 8 to 10 weeks consisted of greater than 99% huMC [23] (link). Unless otherwise stated, experiments were performed in StemPro-34 SFM complete with 100 ng/mL SCF.
5
CD34+ Cell Colony Formation Assay
6
Humanized Mouse Model for Hematopoietic Engraftment
7
Erythroblast Differentiation from UCB CD34+ Cells
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Umbilical cord blood (UCB)-derived CD34+ cells (Stemcell Technologies, 70008.3) were thawed, expanded and differentiated into erythroblasts as described (Douay and Giarratana, 2009 (link); Griffiths et al., 2012 (link)). Briefly CD34+ cells were first expanded for 6 days in ISHI media [IMDM (Gibco), 3U/mL Heparin (Merck), 10 μg/mL Insulin (Sigma), 200 μg/mL of holo-transferrin (Merck) and 5% Human AB serum (Sigma)) in the presence of cytokine cocktail A (60 ng/mL of SCF (Life Technologies), 5 ng/mL of IL3 (Peprotech) and 3U/mL of EPO (R&D Bio-Techne)]. These expanded cells were cryopreserved in batches of 106 cells/mL in a 1:1 mix of ISHI media and freezing media (60% KnockOut Serum Replacement (Gibco), 20% DMSO (Invitrogen) and 20% ISHI media). These “day 6” cells were thawed and used as a starting point for the erythroid differentiation in macrophage co-culture experiments.
8
Culturing Human Leukemic and Colorectal Cancer Cell Lines
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Two human leukemic cell lines, K562 and HL-60, and two human colorectal cancer cell lines, SNU-C4 and HT-29, were obtained from the Korean Cell Line Bank (Seoul National University, Seoul, Korea). All cells were cultured in RPMI1640 or Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Human hematopoietic progenitor CD34+ cells were obtained from STEM CELL Technologies (Vancouver, BC, Canada), and cultured in Hematopoietic Progenitor Expansion Medium DXF with cytokine mix E (PromoCell, Heidelberg, Germany).
9
Isolation of Mononuclear Cells from Blood and Cord Blood
10
Isolation and Co-culture of Human Cardiac Myocytes and CD34+ Cells
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Human adult cardiac myocytes (HACMs) were isolated from the left ventricular tissue obtained from the hearts of patients undergoing heart transplantation. Mechanical dissociation of the tissue and separation of the cardiomyocytes from fibroblasts detached to Petri-dish surface was performed, as described previously
13 (link). All tissue donors gave their informed written consent to the study. The study was approved by the local ethical committee (Medical University of Vienna, Austria; EK 151/2008) and complies with the Declaration of Helsinki.
Human cord blood CD34 positive cells (CD34+ cells) were purchased from StemCell Technologies Company (Grenoble, France). The cells were used in in vitro cell migration assay to assess their migratory capacity toward HACMs. Since porcine CD34+ cells are not commercially available, we used human cardiomyocytes and human cord blood CD34+ cells.
13 (link). All tissue donors gave their informed written consent to the study. The study was approved by the local ethical committee (Medical University of Vienna, Austria; EK 151/2008) and complies with the Declaration of Helsinki.
Human cord blood CD34 positive cells (CD34+ cells) were purchased from StemCell Technologies Company (Grenoble, France). The cells were used in in vitro cell migration assay to assess their migratory capacity toward HACMs. Since porcine CD34+ cells are not commercially available, we used human cardiomyocytes and human cord blood CD34+ cells.
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