A panel of nine fluorophore-conjugated monoclonal antibodies plus a live/dead stain was used for flow cytometric cell sorting and analysis as follows: CD34-PE Cy7 clone 4H11 (eBiosciences); CD38 Alexa Fluor 700 clone HIT2 (eBiosciences); Lineage-APC (containing antibodies against: CD2, CD3, CD14, CD16, CD19, CD56, and CD235a, eBiosciences); CD123-BV605 clone 73G (BD Biosciences); CD45RA-APC eFluor 780 clone HI100 (eBiosciences); CD71-FITC clone OKT9 (eBiosciences); CD41-efluor 450 clone HIP8 (eBiosciences); CD42-PE clone HIP1 (eBiosciences) and CD36 PerCP-Cy5.5 clone CB38 (BD Biosciences). When CD44 was included in the immunophenotyping panel, CD36 PerCP-Cy5.5 was replaced with CD44 PerCP-Cy5.5 clone IM7 (eBiosciences). Cells were stained with specific antibodies for 20 min at 4 °C, and washed prior to staining with Live/Dead Fixable Aqua Dead Stain Kit (Life Technologies) for 30 min at room temperature. Cells were washed and analyzed on a FACSAria III (BD Biosciences) using FACSDIVA™ software v. 8.0.1. Gates were set using strict fluorescence-minus-one controls run for each sample and experiment. Cell doublets, non-viable (AQUA positive), and lineage-positive cells were excluded. Data were analyzed on FlowJo software v. 9.8.2 (Tree Star).
Cd34 pe cy7 clone 4h11
Manufactured by Thermo Fisher Scientific
The CD34-PE Cy7 clone 4H11 is a fluorescent-conjugated antibody used in flow cytometry applications to detect and quantify CD34-positive cells. It targets the CD34 antigen, which is expressed on hematopoietic stem and progenitor cells. This product provides a means to identify and analyze CD34-expressing cell populations.
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2 protocols using cd34 pe cy7 clone 4h11
1
Multiparametric Flow Cytometry Immunophenotyping
2
Isolation and analysis of CD34+ cells from GT-treated patients
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Bone marrow cells from GT-treated patients were obtained in the course of routine follow-up studies of the GT trial and as part of additional exploratory studies. Samples used in these studies were obtained 5 years (FA-02002), 4 years (FA-02004), 3 years (FA-02006), and 2 years (FA-02008) after infusion of transduced CD34 + cells, respectively. Patients FA-02004 and FA-02008 had been treated with eltrombopag to stimulate hematopoiesis 12 and 6 months prior to the evaluation of BM cells, respectively. All samples were processed immediately after BM aspiration. For the purification of CD34 + cells, erythrocytes were lysed with ammonium chloride lysis solution (0.155 mmol/L NH 4 Cl + 0.01 mmol/L KHCO 3 + 10 -4 mmol/L EDTA), washed using PBS (Gibco) + 0.2%BSA (10%) + 2% PenStrep (Gibco) and stained using CD45 APC (clone 2D1; Biolegend) and CD34 PECY7 (clone 4H11; eBiosciences) for 30 minutes at 4°C. DAPI was used at a concentration of 1 μg/mL as a viability marker. CD34 + cells were then sorted in a BD IN-FLUX™ (BD Biosciences) or BD FACSAria II (BD Biosciencs), as previously shown. 2 Purified CD34 + cells were directly used for scRNA seq analysis and small aliquots stored at -80°C for vector copy number analysis.
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