Cells were washed with PBS and then resuspended in binding buffer at a density of 1 × 106 cells/ml. 100 μl of cell suspension was transferred to a 5 ml culture tube, next, 5μl CD138-BV421 and CD34-APC antibody (BD Bioscience, San Diego, CA, USA) were added to the tube. The mixture was vortexed and incubated for 15 min at 4°C in the dark. Finally, the samples were analysed by flow cytometry(BD Bioscience, San Diego, CA, USA).
Cd34 apc antibody
Manufactured by BD
Sourced in United States
The CD34-APC antibody is a laboratory reagent used for the detection and analysis of CD34-positive cells in biological samples. It is conjugated with the fluorescent dye allophycocyanin (APC), which enables the visualization and quantification of CD34-expressing cells through flow cytometry or other fluorescence-based techniques. The core function of this antibody is to specifically bind to the CD34 antigen expressed on the surface of various cell types, including hematopoietic stem and progenitor cells.
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4 protocols using cd34 apc antibody
1
Flow Cytometric Immunophenotyping of Cells
2
Cell-Cycle Analysis of CD34+ HSPC
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To determine the cell-cycle status of healthy CD34+ HSPC after exposure to CM from leukemic cells, CD34+ HSPC were harvested and stained with Hoechst 33342 (Invitrogen, Carlsbad, California), intracellular Ki-67-FITC and a CD34-APC antibody (BD Biosciences, San José, California) (Table S1) for 30 minutes. Cells were subsequently investigated using a MoFlo XDP-High-Speed Cell Sorter (Beckman Coulter GmbH, Krefeld, Germany) and cell-cycle status of CD34+ HSPC was analysed via gating using an analysis software (Summit 5.1, Beckman Coulter GmbH, Krefeld, Deutschland) as previously described. GAPDH served as reference control, and differences in mRNA expression levels were calculated as fold changes by the ΔΔCt method.
3
Evaluating CD34+ AML Cell Survival
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HS‐5 stromal cell feeder layers from shControl and shSPINT2 cells were seeded onto plates (3 × 104 cells/well) in serum‐free RPMI plus BSA and incubated for 48 hours at 37°C. After this period, total bone marrow de novo AML cells were seeded (3 × 105) onto stromal cell monolayers in serum‐free RPMI plus BSA and incubated for another 48 hours at 37°C. Nondherent cells were carefully collected by gentle aspiration, labelled with CD34‐APC antibodies (BD Biosciences), examined by flow cytometry (FACS Calibur), and analysed by FACS Diva software. Percentages of cells expressing the CD34 marker were determined out of a total of 10 000 events.
4
Evaluating CD34+ Cell Adhesion on Stromal Cells
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HS‐5 stromal cells feeder layers from shControl and shSPINT2 cells were seeded in plates (3 × 104 cells/well) in serum‐free RPMI plus BSA and incubated for 48 hours at 37°C. After this period, CD34+ cells isolated from total bone marrow de novo AML cells were seeded (3 × 105) onto stromal cell monolayers for another 24 hours. Nonadherent cells were removed by gentle aspiration, and the CD34+ cells that adhered to HS‐5 stromal cells were collected by gentle pipetting with cold phosphate‐buffered saline (PBS). Cocultured cells were labelled with CD34‐APC antibodies (BD Biosciences), examined by flow cytometry (FACS Calibur) and analysed by FACS Diva software. Cell percentages expressing the CD34 marker were determined out of a total of 10 000 events.
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