HeyA8, HeyA8-MDR, SKOV3, SKOV3-TR, Tyk-nu and Tyk-nu-R cells were harvested using non-enzymatic CellStripper solution (Corning Inc. Corning, NY) to preserve membrane integrity. After washing, the cells (5 × 105) were stained with 10 µL of either FITC-labeled anti-P-gp antibody or PE-labeled anti-MRP1 antibody on ice for 30 minutes. After spinning down, cells were resuspended in HBSS with 1% FBS and expression was analyzed by an LSR II Fortessa cell analyzer (BD Biosciences) (38 (link)). The data were processed by FlowJo software (Tree Star Inc).
Protocol detail
Flow Cytometric Analysis of ABC Transporters
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Anti antibody
Cell
Enzymatic
Fitc
Hbss
Membrane
Mrp1
Tree
Corresponding Organization : University of Chicago
Other organizations : Northeastern University
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Variable analysis
independent variables
- Cell lines: HeyA8, HeyA8-MDR, SKOV3, SKOV3-TR, Tyk-nu and Tyk-nu-R
- Expression of P-gp and MRP1 measured by flow cytometry
- Cell harvesting using non-enzymatic CellStripper solution to preserve membrane integrity
- Cell concentration (5 × 10^5 cells)
- Staining time (30 minutes on ice)
- Staining antibodies (FITC-labeled anti-P-gp, PE-labeled anti-MRP1)
- Flow cytometry analysis using LSR II Fortessa cell analyzer
- Data processing using FlowJo software
- No positive or negative controls were explicitly mentioned in the input protocol.
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