TABLE 3

Experimental details by company (MDCKII-MDR1)

Lab IDCell Source and Passage NumberCulture MediumSeeding DensityCulture TimePlate Format, Material, Pore Size, Vendor Cat. No.Monolayer Integrity Acceptance CriteriaSubstrate and Conc.Assay Buffer and Solvent ContentVolumes AP/BLPreincubationa/Assay timePlate shakingPositive control and Conc.
cells/welldaysnMµlminrpmµM
07Netherlands Cancer Institute p. 13-33DMEM, 10% FBS, 1X NEAA, 1X l-glutamine, 1X Pen-Strep214,0003BD Falcon no. 351181 24-well, PET, 1.0 µmTEER >300 Ωcm23H-Digoxin 39HBSS pH 7.4 +25 mM HEPES 1% DMSO200/70030/9090GF120918 2
04Optivia p. 5-30DMEM, 10% FBS, 1X Pen-Strep150,0003Millipore no. PSRP010R5 24-well, PET, 1.0 µmTEER >90 Ωcm23H-Digoxin 100HBSS pH 7.4 0.5% DMSO250/50030/12060Verapamil 100
06National Cancer Institute p. X - 20DMEM, 10% FBS + 1X l-glutamine, 1X pen-strep, Fungizone175,0007Corning no. 3396 24-well, PC, 0.4 µmLucifer Yellow: <5% transport3H-Digoxin 100HBSS pH 7.4 +10 mM HEPES 1% DMSO100/60030/60NoVerapamil 250
05National Cancer Institute p. 29-36DMEM, 10% FBS52,0004Millipore no. PSRP010R5 24-well, PET, 1.0 µmLucifer Yellow: <0.5 × 10−6 cm/sDigoxin 5000HBSS pH 7.4 +10 mM HEPES 1% DMSO400/10000/60NoGF120918 2
01/02Netherlands Cancer Institute p. 36DMEM + glutamax, 10% FBS, 50 IU/ml penicillin, 50 µg/ml streptomycin160,0003–4BD Falcon no. 351181 24-well, PET, 1.0 µmLucifer Yellow: <5 × 10−6 cm/s3H-Digoxin 120DMEM w/o pyruvate + l-glutamine + 25 mM HEPES + pyridoxine-HCL 1% DMSO450/130030/90∼300GF120918 2
03Netherlands Cancer Institute p. 10 - 20DMEM + glutamax, 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin450,0003Corning no. 3401 12-well, PC, 0.4 µmTEER >150 Ωcm2 Mannitol: <2.0 × 10−6 cm/s3H-Digoxin 50HBSS pH 7.4 +25 mM HEPES <1% DMSO500/180015–30/12060Ketoconazole 100
  • For abbreviations, see Table 1.

  • a Preincubation in presence of inhibitor.