PB-CD34+ cells (4 × 104 cells/mL) were incubated with rhTPO or 2R13 in serum-free expansion medium (SFEM, Stem Cell Technologies, Vancouver, BC, Canada) supplemented with recombinant human stem cell factor (25 ng/mL; PeproTech), recombinant human IL-6 (10 ng/mL; PeproTech), recombinant human IL-9 (10 ng/mL, PeproTech), and LDL (25 μg/mL, Stem Cell Technologies for up to 14 days. Next, the cells were harvested at each time point (days 4, 7, 11, and 14) and labeled with anti-CD41a-FITC-conjugated antibody (Miltenyi Biotec), anti-CD42b-PE-conjugated antibody (Miltenyi Biotec), or 7-amino-actinomycin D (AAD; eBioscience, San Diego, CA, US) at 4 °C for 30 min. After washing with PBS, the cells were analyzed using flow cytometry.
7 amino actinomycin d aad
Manufactured by Thermo Fisher Scientific
Sourced in United States
7-amino-actinomycin D (AAD) is a fluorescent dye used in flow cytometry and other analytical techniques. It binds to DNA, allowing the identification and quantification of cells at different stages of the cell cycle or with different DNA content.
Automatically generated - may contain errors
2 protocols using 7 amino actinomycin d aad
1
Expansion and Differentiation of CD34+ Cells
2
Isolation of Live CD45+ Epidermal Cells
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Single-cell suspensions of digested epidermis samples were incubated with Fc Block (5 mg, rat anti-mouse CD19/CD32, clone 93; eBioscience, San Diego, CA) diluted in phosphate buffered saline for 30 minutes on ice. Samples were subsequently incubated with antigen-presenting celleconjugated rat anti-mouse CD45.2 antibodies (0.5 mg, clone 104; BioLegend, San Diego, CA) diluted in phosphate buffered saline plus 2% serum plus 2 mmol/L EDTA for 30 minutes on ice. Before sorting, cells were labelled with 7-aminoactinomycin D (AAD) (0.25 mg, eBioscience). Live CD45 e cells (7-AAD e and antigen-presenting cell e or PE-Cy7 e ) were sorted using the BD (Franklin Lakes, NJ) ARIA Fusion sorter at 12 p.s.i. with a 100-mm nozzle. Approximately 300,000e500,000 events/cells were collected per sample.
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