Journal: bioRxiv

Article Title: 3D brain angiogenesis model to reconstitute maturation of functional human blood-brain barrier in vitro

doi: 10.1101/471334

Figure Lengend Snippet: (A) 3D confocal image of p-gp protein (red) expressed in tri-cultured vasculature at day 7. Right section view is zoomed from left 3D projection image which describes intraluminal oriented expression of p-gp (pointed as yellow arrow heads). ECs and Nucleus were labeled by anti-CD31 (green) and Hoeschst 33342 (blue), respectively. Scale bar: 20μm. (B) Conceptual description of calcein-AM efflux assay (bottom) which mimics actual effect of p-gp inhibitor on drug resistance by p-gp in vivo (top). (C) Schematic description of work flow for calcein-AM efflux assay in developed in vitro BBB with or without p-gp inhibitor Valsopodar. (D) Representative image of calcein fluorescence in each time point. Vasculature was cultured in EC mono-culture or BBB tri-culture condition with or without p-gp inhibitor pre-treatment. Scale bar: 100μm. (E) Change of calcein fluorescence intensity at the designated region on vessel wall (yellow boxes in 0hour images) every 120 minutes. (F) Quantitative comparison of change in calcein fluorescence intensity during total experiment duration (640 minutes) normalized by initial intensity. (E, F) n=65 for EC only/DMSO Control; n=70 for EC only/Valsopodar treated; n=50 for BBB Tri-culture/DMSO control; n=110 for BBB Tri-culture/Valsopodar treated. Error bars represent SEM. Statistical comparisons between analyzed values of DMSO control and Valsopodar treated group in each time point was obtained from unpaired two-tailed Student’s t-test, with the p value threshold for statistical significance set at *p<0.05; **p<0.005; ***p<0.00001.

Article Snippet: A fully perfused vascular network of EC monoculture or BBB tri-culture was pre-treated with the p-gp inhibitor Valsopodar (PSC-833; Adooq) for 10 hours at a concentration of 10μM.

Techniques: Cell Culture, Expressing, Labeling, In Vivo, In Vitro, Fluorescence, Two Tailed Test

Journal: Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.]

Article Title: Assessment of Drug Transporter Function Using Fluorescent Cell Imaging

doi: 10.1002/0471140856.tx2306s57

Figure Lengend Snippet: Recommended substrates and inhibitors Recommended stock and final concentrations of fluorescent substrate (FS) and positive control inhibitor (I) for each efflux transporter.

Article Snippet: Complete cell culture medium Cell dissociation medium (i.e., 0.25% Trypsin) Dimethylsulfoxide (DMSO) Fluorescent substrates: Rhodamine 123 (Sigma-Aldrich, St. Louis, MO) dissolved in DMSO, 10 mM stock Hoechst 33342 (Sigma-Aldrich, St. Louis, MO), dissolved in deionized water, 10 mM stock Positive control inhibitors: PSC833 (Xenotech, Lenexa, KS), dissolved in DMSO, 1mM stock Ko143 (Sigma-Aldrich, St. Louis, MO), dissolved in DMSO, 1 mM stock Test inhibitors, dissolved in DMSO, 1 mM and 10 mM stock solutions for each chemical Two clear 96-well round bottom microtiter plates with lids (one plate to balance the centrifuge rotor) Microtiter plate centrifuge set to 5°C, 500 × g for 5 min.

Techniques: Positive Control

Journal: Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.]

Article Title: Assessment of Drug Transporter Function Using Fluorescent Cell Imaging

doi: 10.1002/0471140856.tx2306s57

Figure Lengend Snippet: Human embryonic kidney 293 cells stably-transfected with human BCRP or MDR1 genes or empty vector plasmids were incubated with fluorescent substrates (BCRP: Hoechst 33342 and MDR1: Rhodamine 123). Positive inhibitors (BCRP: Ko143 and MDR1: PSC833) were used to block efflux of fluorescent substrates. (A) Line graphs represent the distribution of individual cell fluorescence. Each point represents the mean percent of cells ± SE exhibiting a quantity of fluorescence. (B) Data (bar graphs) are presented as mean relative fluorescence ± SE normalized to cell size.

Article Snippet: Complete cell culture medium Cell dissociation medium (i.e., 0.25% Trypsin) Dimethylsulfoxide (DMSO) Fluorescent substrates: Rhodamine 123 (Sigma-Aldrich, St. Louis, MO) dissolved in DMSO, 10 mM stock Hoechst 33342 (Sigma-Aldrich, St. Louis, MO), dissolved in deionized water, 10 mM stock Positive control inhibitors: PSC833 (Xenotech, Lenexa, KS), dissolved in DMSO, 1mM stock Ko143 (Sigma-Aldrich, St. Louis, MO), dissolved in DMSO, 1 mM stock Test inhibitors, dissolved in DMSO, 1 mM and 10 mM stock solutions for each chemical Two clear 96-well round bottom microtiter plates with lids (one plate to balance the centrifuge rotor) Microtiter plate centrifuge set to 5°C, 500 × g for 5 min.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Incubation, Blocking Assay, Fluorescence