Cell preparation and staining was performed as previously described with minor modifications5 (link). Mononuclear cells (MNCs) were purified by Histopaque 1077 (Sigma-Aldrich) gradient centrifugation. The MNC fraction was washed by centrifugation and resuspension twice in HBSS at room temperature to remove residual platelets. The cell concentration in each sample was then enumerated using an automated cell counter (Beckman Coulter AcT diff hematology analyzer). Five million MNCs were stained for 20 minutes with a cocktail containing saturating amounts of the following mAbs: CD45RA-FITC (Clone ALB11; Beckman Coulter), CD123-PE-Cy7 (Clone 9F5; BD Biosciences), CD38-PerCP-Cy5.5 (Clone HIT2; BD Biosciences), CD34-APC (Clone BIRMA-K3; Dako), CD90-BV421 (Clone 5E10; BD Biosciences), and CD133−PE (Clone AC133; Miltenyi Biotec). Live Dead Aqua (Thermo Fisher) was used to enumerate and exclude dead cells from the analysis. After incubation with mAbs the cells were washed once with PBS containing 0.5% bovine serum albumin, 0.1% sodium azide and 0.0004% tetrasodium EDTA.
Protocol detail
Isolation and Staining of Mononuclear Cells
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Histopaque 1077
AcT diff hematology analyzer
CD45RA-FITC
CD123-PE-Cy7
CD38-PerCP-Cy5.5
CD34-APC
CD90-BV421
CD133−PE
Live Dead Aqua
Bovine serum albumin
Cd123
Cd90
Cells
Centrifugation
Clone
Cy5 5
Edta
Fitc
Hbss
Histopaque
Mabs
Platelets
Sodium azide
Corresponding Organization : University at Buffalo, State University of New York
Other organizations : Roswell Park Comprehensive Cancer Center
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Variable analysis
independent variables
- Cell preparation and staining protocol
- Cell population characteristics (e.g., cell concentration, viability, phenotype)
- Histopaque 1077 gradient centrifugation
- Washing and resuspension in HBSS
- Staining with a cocktail of monoclonal antibodies (mAbs) for 20 minutes
- Positive control: Staining with saturating amounts of CD45RA-FITC, CD123-PE-Cy7, CD38-PerCP-Cy5.5, CD34-APC, CD90-BV421, and CD133-PE mAbs
- Negative control: Live/Dead Aqua staining to exclude dead cells
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