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).
Cytokine mix e
Manufactured by PromoCell
Sourced in Germany
Cytokine mix E is a pre-formulated mixture of cytokines, chemokines, and growth factors. It is designed to support the in vitro cultivation and expansion of various cell types, including immune cells and stem cells. The specific composition and concentration of the components in the mix are optimized to promote cell proliferation, differentiation, and activation.
Automatically generated - may contain errors
Lab products found in correlation
Hematopoietic progenitor expansion medium dxf, by PromoCell (3 mentions)
Cd34 cells, by STEMCELL (2 mentions)
Hl 60, by Korean Cell Line Bank (1 mentions)
Ficoll paque, by Cytiva (1 mentions)
Cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Lymphocyte separation medium, by PromoCell (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
6 protocols using cytokine mix e
1
Culturing Leukemic and Hematopoietic Cells
2
Culturing Human Leukemic Cell Lines
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Human leukemic K562 and HL-60 cell lines 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 were cultured in Hematopoietic Progenitor Expansion Medium DXF with cytokine mix E (PromoCell, Heidelberg, Germany). Human primary leukemic cells were obtained from five patients with AML at the Dong-A University Hospital, Busan, Korea. Informed consent was obtained from all patients before sample collection, according to the Institutional guidelines. Clinical data were obtained by retrospective review of medical records. Bone marrow mononuclear cells were isolated by Ficoll-Paque (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) density-gradient centrifugation. Samples ranged from 75% to 91% blasts. Flow cytometry immunophenotyping using CD45 dim (+), lineage-specific (myeloid) markers, and side scatter (SSC) confirmed that cell sample purity exceeded 95%. These cells were cultured in RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cultures were maintained in a humidified atmosphere of 95% air/5% CO2 at 37°C.
3
Generation of Monocytes from CD34+ Stem Cells
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For in vitro generation of monocytes, haematopoietic CD34+ stem cells were isolated and monocytes generated according to our standardized protocol (manuscript in preparation). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from EDTA anticoagulated blood by Ficoll-Paque (Lymphocyte Separation Medium; PAA, Cölbe, Germany) gradient density centrifugation. CD34+ cells were isolated using the CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). For determining quality of isolation, CD34+ cells were stained with anti-CD34 APC (581; BD Biosciences) and anti-CD45 PE (HI30; BD Biosciences) antibodies. Mean purity assessed by flow cytometry was 85.3 ± 4.8%.
Isolated CD34+ haematopoietic stem cells were expanded in 6-well plates (1 × 104 cells/mL) for 13 days in the Haematopoietic Progenitor Cell Expansion Medium DXF, enriched with Cytokine Mix E (PromoCell GmbH, Heidelberg, Germany). After expansion, cells were seeded in 12-well plates for differentiation to monocytes in the Haematopoietic Progenitor Medium (PromoCell GmbH) supplemented with iron preparations. Daily progress was flow cytometrically monitored after anti-CD14, anti-CD16 and anti-CD86 staining, subdividing cells into monocyte subsets. At Day 7 of differentiation, surface expression of CCR5 and CX3CR1 was additionally measured.
Isolated CD34+ haematopoietic stem cells were expanded in 6-well plates (1 × 104 cells/mL) for 13 days in the Haematopoietic Progenitor Cell Expansion Medium DXF, enriched with Cytokine Mix E (PromoCell GmbH, Heidelberg, Germany). After expansion, cells were seeded in 12-well plates for differentiation to monocytes in the Haematopoietic Progenitor Medium (PromoCell GmbH) supplemented with iron preparations. Daily progress was flow cytometrically monitored after anti-CD14, anti-CD16 and anti-CD86 staining, subdividing cells into monocyte subsets. At Day 7 of differentiation, surface expression of CCR5 and CX3CR1 was additionally measured.
4
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).
5
Cytotoxicity Evaluation of AZA and Teriflunomide
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U937, HL-60, R-U937, and R-HL-60 cells and leukemia cells from patients were incubated in RPMI1640 medium (Life Technologies Inc., Carlsbad, CA, USA) including 10% inactivated fetal bovine serum and 1% penicillin/streptomycin (Life Technologies). CD34-positive cells from a healthy donor was cultured in Hematopoietic Progenitor Expansion Medium DXF (PromoCell, Heidelberg, Germany) supplemented with Cytokine Mix E (PromoCell). For treatment with reagents, cells were collected by centrifugation and suspended at 1 × 105 cells/ml in fresh medium with the agents or 0.01% DMSO as the vehicle. Cell viability was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) as previously described [25 (link)]. CI 25 was calculated using Chou-Talalay method [14 (link)]. For continuous treatment, 5 μM AZA and/or 1 μM teriflunomide was added to the U937 cells or HL-60 cells suspended in the fresh medium at 1 × 105 cells/ml at day 0. Then at days 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42, and 49, culture medium was changed to fresh medium with the added agents and cell viability was measured using the Cell Counting Kit-8.
6
Isolation and Maintenance of Human Cord Blood Hematopoietic Stem and Progenitor Cells
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Human umbilical cord blood was obtained from DKMS cord blood bank gGmbH (Dresden, Germany) or the cord blood bank of the German Red Cross Services (Mannheim, Germany) after informed consent of the parents and approval by the local ethics committee (Ethik-Kommission der Landesärztekammer Baden-Württemberg, project number B-F-2013-111). Mononuclear cells were isolated from cord blood samples up to 48 h after collection by density gradient centrifugation using lymphocyte separation medium (PromoCell GmbH, Heidelberg, Germany). Subsequently, HSPCs were obtained from the mononuclear cell fraction by positive selection for the cell surface marker CD34 via magnetic activated cell sorting (MACS, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. HSPCs were used for experiments if the CD34+ cell fraction was at least 95% as determined by flow cytometry. HSPCs were maintained in Hematopoietic Progenitor Expansion Medium DXF (PromoCell GmbH, Heidelberg, Germany) with 1% of Cytokine Mix E (PromoCell GmbH, Heidelberg, Germany) in a humidified incubator at 37°C and 5% CO2.
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