Human CD34+ HSPC were isolated from bone marrow mononuclear cell fraction using Ficoll gradient centrifugation followed by magnetic bead separation using Human CD34 Progenitor Cell Isolation Kit, (Miltenyi Biotech, #130-046-703). CD34+ cells were cultured in a density of 2 x 105 cells/mL in Stemline II Hematopoietic Stem Cell Expansion medium (Sigma Aldrich, #50192) supplemented with 10 % FBS, 1 % penicillin/streptomycin, 1 % L-Glutamine and a cytokine cocktail consisting of 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL TPO, 50 ng/ml SCF and 50 ng/mL FLT-3L (all cytokines were purchased from R&D Systems). Human induced pluripotent stem cells (iPSC) were cultured on Geltrex LDEV-free reduced growth factor basement membrane matrix (Thermo Fisher Scientific, #A1413201) coated plates in a density of 2 x 105 cells/mL in StemFlex medium (Thermo Fisher Scientific, #A3349401) supplemented with 1 % penicillin/streptomycin. HL60 cells were maintained in RPMI-1640 supplemented with 10 % fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA, USA), 2 mM L-glutamine, and 1 % penicillin/streptomycin (Thermo Fisher Scientific) at 37°C and 5 % CO2.
Human cd34 progenitor cell isolation kit
Manufactured by Miltenyi Biotec
The Human CD34 Progenitor Cell Isolation Kit is a laboratory equipment product that enables the isolation of human CD34-positive progenitor cells from various sample types, such as peripheral blood, bone marrow, or cord blood. The kit utilizes magnetic bead technology to selectively enrich for the target cell population.
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Lab products found in correlation
Facsaria sorter, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Stemflex medium, by Thermo Fisher Scientific (1 mentions)
Flt3l, by R&D Systems (1 mentions)
Stemline 2 hematopoietic stem cell expansion medium, by Merck Group (1 mentions)
Geltrex ldev free reduced growth factor basement membrane matrix, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by R&D Systems (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Rpmi 1640, by Gemini Bio (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Interleukin 3 (il 3), by R&D Systems (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Anti cd38 fitc, by BD (1 mentions)
Facscanto 2 equipment, by BD (1 mentions)
Anti cd11c fitc, by BD (1 mentions)
Anti cd56 pe, by BD (1 mentions)
Anti cd34 apc, by BD (1 mentions)
Anti cd16 apc, by BD (1 mentions)
4 protocols using human cd34 progenitor cell isolation kit
1
Isolation and Culture of Human HSPCs and iPSCs
2
Isolation of CD34+ Hematopoietic Stem Cells
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Mononuclear cells (MNC) from umbilical cord blood (UCB) were prepared by Ficoll-Paque Plus (GE Healthcare Bioscience) gradient separation. CD34+ cells containing hematopoietic stem and progenitor cells were enriched from the MNC fraction using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to manufacturer instructions, with final purity frequencies between 85 and 95%. Cells were enumerated before analyses, and the purity confirmed by flow cytometry.
3
Isolation and Characterization of Hematopoietic Stem and Progenitor Cells
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CD34+ cells from normal bone marrow (NBM) and ALL were enriched using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. After staining with PE-conjugated anti-lineage markers (CD3, CD8, TCR, CD56, CD14, CD11b, CD20, CD19, and CD235a) and anti-CD34-APC, primitive cell populations were highly purified by multicolor flow cytometry using a FACSAria sorter (BD Biosciences). HSPC were separated as Lin-CD34+. Upon harvesting from culture, anti-CD56 and anti-CD19 antibodies were used to evaluate cell production in lymphoid lineage conditions. Pro-B blast population was identified as CD34+CD19+, while Pre-B cells as CD34−CD19+.
4
Purification and Characterization of Primitive Blood Cell Populations
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CD34+ cells from ABM and UCB were enriched using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to manufacturer instructions and our previous reports [9 (link)]. After staining with PE-conjugated antilineage markers (CD3, CD8, TCR, CD56, CD14, CD11b, CD20, CD19, and CD235a), anti-CD34-APC, anti-CD38-FITC, and anti-CD45RA-PE-TxR conjugated antibodies, primitive cell populations were highly purified by multicolor flow cytometry using a FACSAria sorter (BD Biosciences). Hematopoietic stem cells (HSC) were separated as Lin−CD34+CD38−CD45RA−, while multipotent progenitors (MPP) were sorted as Lin−CD34+CD38+CD45RA−, and early lymphoid progenitors (ELP) as Lin−CD34+CD38+CD45RA+, as described [9 (link)]. Upon harvesting from culture, anti-CD56-PE, anti-CD11c-FITC, and anti-CD16-APC (BD Biosciences) were used to evaluate innate cell production in lymphoid lineage conditions. NK cells were identified by flow cytometry in a FACSCanto II equipment (BD Biosciences) as CD56+CD11c+ or CD56+CD16+CD11c+ cells, while dendritic cells (DC) were detected as CD56−CD11c+. Analysis of flow cytometry data was performed using the FlowJo 10 software (TreeStar Inc., USA).
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