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).
Cd34 antibody
Manufactured by BioLegend
Sourced in United States
The CD34 antibody is a laboratory reagent used to detect and quantify the presence of CD34 protein in biological samples. CD34 is a transmembrane glycoprotein expressed on the surface of hematopoietic stem and progenitor cells, as well as on the endothelial cells of blood vessels. This antibody can be used in various immunoassay techniques to identify and study these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Hanks balanced salt solution (hbss), by Beyotime (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Ficoll paque plus solution, by GE Healthcare (1 mentions)
Ficoll paque plus, by Miltenyi Biotec (1 mentions)
Anti cd34 coated magnetic beads, by Miltenyi Biotec (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Cd34 fitc, by BioLegend (1 mentions)
Cd34 microbeads kit, by Miltenyi Biotec (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fortessa, by BD (1 mentions)
4 protocols using cd34 antibody
1
Isolation of AML and Healthy HSPCs
2
CD34+ Blast Enrichment and Purification
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Blood cells from patient samples were enriched for CD34-positive blasts by using immunomagnetic beads according to the manufacturer's protocol (CD34 MicroBeads Kit; Miltenyi Biotec; #130-046-702). Purity was controlled using flow cytometry (FACS) against the precursor antigen CD34 (CD34-antibody conjugated with fluorescein-isothiocyanate (CD34-Fitc), Lot B213405, monoclonal, mouse anti-human; Biolegend) (24 (link)). CD34-positive blasts were used for cell-panning directly after sorting. If the initial blast cell count after DGC was below 75%, all three selection rounds were conducted on CD34-enriched material. If cell counts were above 90%, no CD34 enrichment was performed. Cells were only purified for the first SR if blast counts were between 75 and 90%.
3
Cell Surface Marker Profiling
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Reprogramming cells at different days were digested by 0.25% trypsin, and wash with PBS. The cells were resuspension with flow cytometry buffer (PBS with 2% FBS), and CD34 antibody (BioLegend, 119307) or CD104 antibody (BioLegend, 123607) was used as recommended by the manufacturer for 15 min at 4 C. After staining, cells were washed by flow cytometry buffer and resuspension with flow cytometry buffer containing DAPI (2.5 mg/mL, Sigma), and analyzed with Fortessa (BD Biosciences) or sorted with Arial (BD Biosciences). The FACS data was analysis with FlowJo software.
4
Immunostaining of Reprogrammed Cells
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Reprogramming cells were changed with fresh medium containing CD34 antibody (BioLegend, 119307) at a final concentration of 2 mg/mL. The cells were kept under normal culture conditions in an incubator for one hour, and then the medium was removed. Cells were washed with DPBS twice, and then the medium was changed to fresh medium. The light was avoided during this process. After staining, cells were scanned in a BiosStation CT.
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