For immunophenotyping of isolated progenitor cells and to monitor evolution upon amplification, flow cytometry was performed with a FACS CaliburTM (Beckton Dickinson, 11, rue Aristide Bergès, ZI des Iles, BP4, 38801 Le Pont de Claix Cedex, France) cytometer. The different phases of the cell cycle were observed after propidium iodide labeling as described [40 (link)], and the flow cytometry histograms were analyzed with the Modfit LT v3.2 software (Verity Software House, Topsham, ME, USA). Cells were labelled with VioBlue-conjugated anti-CD34 antibody, phycoerythrin (PE)-conjugated anti-CD38 antibody, and a lineage mix of fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD19, CD56, CD13, CD14, and CD11a antibodies (Miltenyi Biotech). For differentiation assay, a complementary labelling was carried out with Brilliant Violet-conjugated anti-CD34 (Beckton Dickinson Pharmingen), FITC-conjugated anti-CD36 (Beckman Coulter, Villepinte, France), PE-conjugated anti-CD235a (GPA) (Beckman Coulter), and allophycocyanin (APC)-conjugated anti-CD71 (Beckman Coulter) antibodies.
Vioblue conjugated anti cd34 antibody
Manufactured by Miltenyi Biotec
The VioBlue-conjugated anti-CD34 antibody is a laboratory reagent used for the detection and analysis of CD34-positive cells. CD34 is a surface marker expressed on hematopoietic stem and progenitor cells. The VioBlue fluorescent dye is conjugated to the anti-CD34 antibody, allowing for the identification and enumeration of CD34-positive cells using flow cytometry or other fluorescence-based techniques.
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2 protocols using vioblue conjugated anti cd34 antibody
1
Immunophenotyping of Progenitor Cells
2
Phenotypic and Transduction Efficiency of Stem Cells and T Cells
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The phenotype and efficiency of transduction of hCD34+ HSPCs and T lymphocytes were analyzed by Flow Cytometry (FACS Canto II Instrument and DIVA software). hCD34+ HSPCs were stained with PE-conjugated anti-CD90 antibody (BD PharMingen), VioBlue-conjugated anti-CD34 antibody (Miltenyi Biotec), and antigen-presenting cell (APC)-conjugated anti-CD133 antibody (Miltenyi Biotec).
PBMCs were stained with anti-CD3 monoclonal antibody before and after activation (Miltenyi Biotec). In selected experiments, T cell memory phenotype was detected by staining with monoclonal antibodies specific for CD3 (VioGreen-conjugated), CD8 (APC-Vio770-conjugated), CD45RA (PE-Vio770-conjugated), CD62L (VioBlue-conjugated), and CD95 (APC-conjugated) (Miltenyi Biotec). For both cell types, cell mortality and transduction efficiency were evaluated by 7-AAD labeling (BD PharMingen) and by the level of GFP expression, respectively.
PBMCs were stained with anti-CD3 monoclonal antibody before and after activation (Miltenyi Biotec). In selected experiments, T cell memory phenotype was detected by staining with monoclonal antibodies specific for CD3 (VioGreen-conjugated), CD8 (APC-Vio770-conjugated), CD45RA (PE-Vio770-conjugated), CD62L (VioBlue-conjugated), and CD95 (APC-conjugated) (Miltenyi Biotec). For both cell types, cell mortality and transduction efficiency were evaluated by 7-AAD labeling (BD PharMingen) and by the level of GFP expression, respectively.
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