Measuring P-gp Functionality in iPS-ECs

P-gp functionality was assessed using rhodamine 123 (Sigma), a substrate for P-gp, and efflux transporter inhibitors as follows. Human iPS-ECs, which were co-cultured with C6 cells or incubated in C6CM, were washed with PBS and pre-incubated at 37°C for 1 hr with or without inhibitors (5 μM cyclosporine A (Wako) or 10 μM MK571 (Sigma)), followed by addition of rhodamine 123 to the upper chamber. After 1 hr, the fluorescence activity of the medium in the lower chamber was quantified on a plate reader [24 (link)].

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Minami H., Tashiro K., Okada A., Hirata N., Yamaguchi T., Takayama K., Mizuguchi H, & Kawabata K. (2015). Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells. PLoS ONE, 10(6), e0128890.

Publication 2015

rhodamine 123 cyclosporine A MK571

Cultured cells Cyclosporine a Fluorescence Human Inhibitors Rhodamine 123 Transporter

Corresponding Organization :

Other organizations : Osaka University, National Institute of Biomedical Innovation, Health and Nutrition

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Variable analysis

independent variables

Presence or absence of inhibitors (5 μM cyclosporine A or 10 μM MK571)

dependent variables

Fluorescence activity of the medium in the lower chamber

control variables

Cell type (human iPS-ECs co-cultured with C6 cells or incubated in C6CM)
Incubation time (1 hr pre-incubation with or without inhibitors, followed by 1 hr incubation with rhodamine 123)

controls

Positive control: Incubation with rhodamine 123 without inhibitors
Negative control: Not explicitly mentioned

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