Three milliliters of EDTA PB samples (all 211 subjects) or 1 mL of EDTA BM sample (only 84 subjects) were centrally analyzed at Flow‐cytometry lab in Siena within 24 h of collection, since in preliminary experiments it was shown that within 24 h cell viability was superior to 80%. BM and PB leukocytes and red blood cells count was performed using a Unicell DxH 800 Coulter (Beckman Coulter, Brea, CA). In all PB and BM samples, the presence of CD34+/CD38−/CD26+ population was evaluated by multiparametric flow cytometry analysis using a four‐color staining standardized protocol with lyse stain wash procedure. Red cells’ lysis was performed with BD Pharm Lyse™ ammonium chloride (Ref 555899, BD Biosciences, San Jose, CA), 1:10 diluted in deionized water, using BD FACS™ Lyse Wash Assistant (LWA) instrument (BD Biosciences, San Jose, CA). After lysis, 2.0 × 106 leucocytes per mL were incubated with a custom‐made lyophilized pre‐titrated antibody mixture test and control tube (Ref 625183, BD Biosciences, San Jose, CA), containing CD34‐FITC (clone 581), CD26‐PE (clone M‐A261), CD38‐APC (clone HIT2), and CD45‐V500 (clone 2D1) for tube test and CD34‐FITC (clone 581), anti IGg1 PE, CD38‐APC (clone HIT2), and CD45‐V500 (clone 2D1) for control tube.
Cd26 pe clone m a261
Manufactured by BD
CD26‐PE (clone M‐A261) is a fluorescently-labeled monoclonal antibody that binds to the CD26 cell surface antigen. CD26 is a transmembrane glycoprotein expressed on various cell types. The PE (Phycoerythrin) fluorescent label allows for the detection and analysis of CD26-positive cells by flow cytometry.
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2 protocols using cd26 pe clone m a261
1
Multiparametric Flow Cytometry of CD34+/CD38-/CD26+ Cells
2
CD26 Expression in Bone Marrow Progenitors
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For each patient 2 mL of bone marrow samples collected in EDTA tubes were analyzed. CD26 expression was assessed by multiparametric flow cytometry, analyzing the CD45+/CD34+/CD38− population using a four-color protocol, as previously reported by our group (15 (link)). Briefly, 2.0 x 106 leucocytes were incubated with BD Pharmigen CD45-V500 (clone 2D1), CD34-FITC (clone 581), CD38-APC (clone HIT2), CD26-PE (clone M-A261), and proper negative controls. After washing, acquisition and analysis of at least 1.0 × 106 of CD45+ cells were performed by using a MACSQuant Analyzer (Miltenyi, Gemany), equipped with 3 lasers and 8 fluorescent channels available. The absolute number of CD26+ cells was calculated by multiplying the number of white cells/μL automatically counted for the proportion of CD34+/CD38−/CD26+ on CD45+ cells using the MACSQuantify Software (Miltenyi).
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