The detached cells were collected by centrifugation, aliquoted at 500,000 cells/tube, and incubated with the test compounds at the indicated concentrations and calcein AM or MitoTrackerH Green FM for 10 minutes in a 37uC water bath. After incubation, the cells were collected by centrifugation at 800xg for 5 minutes and resus pended in PBS/0. 1% BSA. The fluorescent substrates retained in KB V1 cells were detected by flow cytometry. The data were analyzed with FlowJo 7. 2. 2 and presented as histograms. The percentage of the maximum is the number of cells in each bin divided by the number of cells in the bin that contains the largest number of cells. Total number of cells counted per sample was 10,000.
Photolabeling of ABCB1 with IAAP The interaction of selected compounds with ABCB1 was assessed by an in vitro photolabeling assay as previously described. Crude membranes from Hifive cells overexpressing human ABCB1 were incubated with the selected compounds for 5 minutes, after which 3 to 5 nmol/L IAAP in 50 mM Tris HCl was added. After exposure to UV light selleck inhibitor for 10 minutes at room temperature to covalently crosslink IAAP with ABCB1, the samples were separated by electro phoresis, and gels were dried and exposed on Bio Max MR film. Cell viability assay HCT 15 Pgp cells were plated and cultured in 96 well plates for 1 or 2 days, until they reached 50 80% confluency. The cells were treated with the indicated concentrations of BI 2536, cyclosporin A, and XR9576 for 48 hours.
Cell viability was examined using selelck kinase inhibitor the Cell Counting Kit 8, which functions similar to MTT assays by reduction of the tetrazolium salt, WST 8, to yield a yellow colored and water soluble formazan dye. Data analysis The IncuCyteTMFLR software provides the mean of the total fluorescence intensity, obtained by averaging fluorescent intensities from all the pixels in the image from each well. The automatically generated mean fluorescence intensity is not corrected for the background fluorescence, which can often lead to distorted outputs. Additional software, the Object Counting v2. 0 Analysis software provided by the IncuCy teTMFLR, can assist further quantification and analysis of the fluorescent images.
Using this software, a segmentation mask was generated to separate fluorescent and non fluorescent objects so that the sum of the fluorescent intensity of the positive cells can be calculated without the interference from the background fluorescence.

The Object Summed Intensity per mm2 from the metrix menu was chosen to indicate the background corrected fluorescent intensity of each well and designated as the object intensity. The relative inhibition of each compound on calcein AM efflux was calculated by the following equation: %inhibition 100 XT{XcalceinAM eT XMax or XR9576{XcalceinAM eT, where X represents the mean fluorescence intensities or the object intensities, and T denotes the test compound.