Assays were performed in DPBS-H (10 mM HEPES, 25 mM glucose, in Dulbecco's phosphate-buffered saline with calcium chloride and magnesium chloride) in 24-well transwell plates on a rocking shaker at 20 rpm, 37 °C, 95% humidity, and 5% CO2. All substrates were dissolved at specific concentrations in DPBS-H and added to the luminal (A) or abluminal (B) side. The incubation times for the substrate were 15, 30, 45, and 60 min. The concentrations of the substrates were measured by a fluorescence microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific). Digoxin, dantrolene, and salazosulfapyridine samples were pretreated with acetonitrile precipitation of proteins and measured using an LC-MS/MS system (ExionLC-QTRAP6500+, SCIEX, Framingham, Massachusetts, USA). The compound concentrations were as follows: rhodamine 123 (10 μM), Hoechst 33,342 (200 μM), 2-NBDG (100 μg/ml), digoxin (5 μM), dantrolene (5 μM), and salazosulfapyridine (sulfasalazine, SASP) (5 μM).
In this study, we employed Pe as the permeability coefficient because researchers can eliminate the influence of the insert membranes. The permeability coefficient (Pe) was calculated according to Nakagawa et al. [16 (link)] by dividing the amount of substrate in the luminal compartment (A) by the substrate concentration in the abluminal compartment (B). The volume was obtained at multiple timepoints according to the following formula: where [C]r is the amount of the compound in the receiving compartment, [C]d represents the amount of the compound in the donor compartment, and [R] is the volume of the receiving compartment. When the volume is plotted over time, the slope equals the permeability × surface area product (PS) of the membrane. The PS of the membrane with cells is called the total PS (PStotal), and the PS of the membrane without cells is called the membrane PS (PSmem). The Pe can be computed from the PStotal and PSmem:
1/PSe = 1/PStotal-1/PSmem where the units of PS and surface area are μL/mL and cm2, respectively.
To calculate Pe (cm/min), the PSe value was divided by the surface area (S) of the membrane:
In this study, we employed Pe as the permeability coefficient because researchers can eliminate the influence of the insert membranes. The permeability coefficient (Pe) was calculated according to Nakagawa et al. [16 (link)] by dividing the amount of substrate in the luminal compartment (A) by the substrate concentration in the abluminal compartment (B). The volume was obtained at multiple timepoints according to the following formula: where [C]r is the amount of the compound in the receiving compartment, [C]d represents the amount of the compound in the donor compartment, and [R] is the volume of the receiving compartment. When the volume is plotted over time, the slope equals the permeability × surface area product (PS) of the membrane. The PS of the membrane with cells is called the total PS (PStotal), and the PS of the membrane without cells is called the membrane PS (PSmem). The Pe can be computed from the PStotal and PSmem:
1/PSe = 1/PStotal-1/PSmem where the units of PS and surface area are μL/mL and cm2, respectively.
To calculate Pe (cm/min), the PSe value was divided by the surface area (S) of the membrane:
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