PE-Cy™7 Mouse Anti-Human CD123

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To perform flow cytometry analysis and to sort BFU-E and CFU-E cells from primary human samples including BM, cord blood, and peripheral blood, we isolated CD45+ cells from mononuclear cells (MNC) derived by Ficoll density gradient separation, followed by positive selection using CD45 magnetic beads.	Ab incubation for 30 minutes in the dark. Cells were washed twice with 40 mL phosphate-buffered saline/0.5% bovine serum albumin, resuspended in 5 mL phosphate-buffered saline/0.5% bovine serum albumin, and stained with the viability marker 7-AAD on ice for 10 minutes in the dark.	The analysis was performed using BD FACSDiva. Sorting was performed using a MoFlo high-speed cell sorter (Beckman Coulter).

Upstream tips
To perform flow cytometry analysis and to sort BFU-E and CFU-E cells from primary human samples including BM, cord blood, and peripheral blood, we isolated CD45+ cells from mononuclear cells (MNC) derived by Ficoll density gradient separation, followed by positive selection using CD45 magnetic beads.
Protocol tips
Ab incubation for 30 minutes in the dark. Cells were washed twice with 40 mL phosphate-buffered saline/0.5% bovine serum albumin, resuspended in 5 mL phosphate-buffered saline/0.5% bovine serum albumin, and stained with the viability marker 7-AAD on ice for 10 minutes in the dark.
Downstream tips
The analysis was performed using BD FACSDiva. Sorting was performed using a MoFlo high-speed cell sorter (Beckman Coulter).

Publication protocol

To perform flow cytometry analysis and to sort BFU-E and CFU-E cells from primary human samples including BM, cord blood, and peripheral blood, we isolated CD45+ cells from mononuclear cells (MNC) derived by Ficoll density gradient separation, followed by positive selection using CD45 magnetic beads. The CD45+ cells were stained with the following mouse anti-human antibodies: PE-conjugated anti-CD34, fluorescein isothiocyanate-conjugated anti-CD36, PE-Cy7–conjugated anti–IL-3R (CD123), V450-conjugated anti-CD45, APC-conjugated anti-CD41 and anti-GPA, PerCp-conjugated anti-CD3, anti-CD4, anti-CD14, and anti-CD19, APC-H7–conjugated anti-CD71, or appropriate isotype controls (all from BD Biosciences) for 30 minutes in the dark. Cells were washed twice with 40 mL phosphate-buffered saline/0.5% bovine serum albumin, resuspended in 5 mL phosphate-buffered saline/0.5% bovine serum albumin, and stained with the viability marker 7-AAD on ice for 10 minutes in the dark. The analysis was performed using BD FACSDiva. Sorting was performed using a MoFlo high-speed cell sorter (Beckman Coulter). The number of BFU-E or CFU-E cells in one million MNCs was quantitated.

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