After enrichment, cells were washed and incubated at a dilution of 1/50 with mouse serum (Sigma-Aldrich) for 10 min at 4 °C. Cells were stained with anti-CD3 (clone UCHT1, FITC, Beckman Coulter), anti-CD14 (clone RMO52, FITC, Beckman Coulter), anti-CD19 (clone J3-119, FITC, Beckman Coulter) anti-CD45 (clone HI30, APC-Cy7, Biolegend) and anti-CD56 (clone N901(NKH-1), PE, Beckman Coulter) antibodies for 30 min at 4 °C. We added an anti-CD34 (clone 581, FITC, BD Biosciences) antibody to the lineage to sort AML samples. Cells were then washed twice with DPBS and incubated in DPBS with a dead cell marker for 10 min at 4 °C. Cells were washed again and sorted in an Influx or Melody cell sorter from BD (Becton Dickinson, San Diego, USA).
Anti cd34 clone 581
Manufactured by BD
Anti-CD34 (clone 581) is a monoclonal antibody that specifically binds to the CD34 antigen. CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells. The antibody clone 581 is used for the identification and enumeration of CD34-positive cells in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Melody cell sorter, by BD (1 mentions)
Mouse serum, by Merck Group (1 mentions)
Anti cd3 clone ucht1, by Beckman Coulter (1 mentions)
Facsaria 2, by BD (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Aria 2, by BD (1 mentions)
Histopaque 1083, by Merck Group (1 mentions)
Anti cd8a clone 53 6, by Thermo Fisher Scientific (1 mentions)
2 protocols using anti cd34 clone 581
1
Multicolor Flow Cytometry for Immune Cell Characterization
2
Isolation and Sorting of Murine and Human Hematopoietic Stem Cells
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Low‐density mononuclear cells were isolated by density centrifugation (Histopaque 1083, Sigma) and stained with a cocktail of biotinylated lineage antibodies all rat anti‐mouse antibodies: anti‐CD11b (clone M1/70), anti‐B220 (clone RA3‐6B2), anti‐CD5 (clone 53‐7.3) anti‐Gr‐1 (clone RB6‐8C5), anti‐Ter119 and anti‐CD8a (clone 53‐6.7; eBioscience). After lineage depletion by magnetic separation (Dynabeads, Invitrogen), cells were stained with anti‐Sca‐1 (clone D7), anti‐c‐Kit (clone 2B8), anti‐CD34 (clone RAM34), anti‐Flk2 (clone A2F10), and Streptavidin antibodies (eBioscience). Stained HSCs (gated as Lin−Sca+Kit+CD34−Flk2−) were sorted in 96 well plates using a BD FACS Aria II (BD Bioscience) as previously reported (Florian et al, 2012 (link)). Human BM MNCs were enriched using standard Ficoll‐based density centrifugation in the clinical laboratory at CCHMC. Human HSPCs (linneg CD34pos SSClow cells from BM) were FACS‐sorted (BD Aria II, BD Bioscience) according to standard protocols (sorted linneg CD34pos SSClow cells from BM, anti‐CD34 clone 581 (BD #555824)), human hematopoietic lineage antibody cocktail, FITC, eBioscience (#22‐7778‐72; Kumar et al, 2021 (link)).
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