| Assay Detail |
Assay materials used were as described by Di et. Al.(2) with minor deviations. Fetal Bovine Serum (FBS) and L-Glutamine 200 mM were purchased from Corning Inc. (Corning, NY). Penicillin-Streptomycin (Pen/Strep) was purchased from the Lonza Group (Basel, Switzerland). Hanks Balanced Salt Solution (HBSS), HEPES, and Calcium Chloride (CaCl2) were purchased from Fisher Scientific (Hampton, NH). 1M Anhydrous Dextrose was used in place of 1M D-Glucose and was purchased from EMD Millipore (Ontario, Canada). Magnesium Chloride Hexahydrate was used in place of Magnesium Chloride and was purchased from Sigma-Aldritch (St. Louis, MO). The MEM-α media and Buffer B solution were delivered with a 12-Channel pipette. Cell culturing was performed as described by Di et. Al.(2) with minor modifications. MDCKII-LE cells were cultured at 37°C, 5% CO2, 95% relative humidity in T25 flasks prior to splitting which took place between 3-5 days or when cells reach approximately 90% confluency. Under sterile conditions in a biological safety cabinet, media were removed from the T25 flask containing the cells, followed by two 5mL rinses with 0.5mL of 0.25% Trypsin (prewarmed to 37°C) evenly across the bottom of the flask. The flasks were incubated for 2-5 minutes, or until cells no longer adhered to the flask. Four mL of MEM-alpha were added to the flask to dilute the trypsin and quench proteolysis. This was followed by appropriate dilution of cells into new flasks (T25/T50/T75) to continue the cell line.The cells were typically cultured in one or two T75 flasks when prepared for the assay. Media was aspirated, followed by spreading 1.5mL of 0.25% Trypsin and incubation for 2-5 minutes or until cells dislodged from the surface of the flask. 8.5mL of MEM-alpha media was added to the trypsinized cells to get a total of 10mL MEM-alpha. Cells were counted using a hemocytometer and diluted to a volume of 5 x 10^5 cells/mL in complete MEM-alpha media. Cells were then plated using the same methods as Di et al(2). The seeded plates were left in the incubator for 5 days. On the day of the permeability assay, media of assay plates is removed, and plates are rinsed with Buffer B. Lucifer Yellow (LY) (excitation wavelength: 425-430 nm1; emission wavelength: 515-520nm(3, 4)) was incorporated into the assay to provide a rapid measure of monolayer quality. A 15mM stock of LY was prepared in Buffer B a day prior to the assay. From this stock, a 15uM stock was prepared, followed by a serial 1:3 dilution with Buffer B into a 96-well plate—giving a range from 15uM to 20.5nM with the final well containing only Buffer B. The series was repeated in triplicate and read in a PerkinElmer Envision Plate Reader, alongside 4-5 wells containing the 5uM LY in Buffer B stock used in the assay.
Sample compound mixtures were prepared as described above in a 2 mM stock in DMSO, delivering 10 uL into a 96-well conical plate. 90 uL of Buffer B with 5 uM LY was added to the 10 uL DMSO stock to yield a 200 uM compound concentration in 10% DMSO, with thorough mixing (precipitation is evident at this concentration for some library mixtures). 10 uL of this mixture was diluted with 990 uL of Buffer B with 5 uM LY, with thorough mixing, for a final concentration of 2 uM compound and 0.1% DMSO, as the “t=0” dilution.An initial fluorescence reading of the “t=0” dilution was collected as LYt=0. 75uL of the final dilution was added to the apical layer of the assay plates. 250uL of Buffer B (without LY) was added to the basal/collector plate. The assay plate was incubated for 90 minutes then separated. 100uL of the basal layer was separated for a fluorescence reading as LYt=90. 50uL of the basal layer was transferred to a 96-well conical plate, mixed with 50uL of acetonitrile, and sealed with a pierceable seal for UPLC-MS quantification (Ibasal) . The same process was performed for the “t=0” dilution before application to the cell monolayer (It=0). Protein contamination in the basal layer collected on the column was backflushed from the column after analyzing plates (30:30:30:10 MeCN:MeOH:iPrOH:H2O). Permeability rates across the cell monolayer were calculated via the following equation: Papp = dMr/dt * 1/(Msa*CD(0)) [6]. Where:dMr/dt = flux of the compound across the cell monolayer. Msa = surface area of filter membrane = 0.11 cm^2. CD(0) = concentration of compound in the donor well at “t=0”.
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