Lab ID | Cell Source and Passage Number | Culture Medium | Seeding Density | Culture Time | Plate Format, Material, Pore Size, Vendor Cat. No. | Monolayer Integrity Acceptance Criteria | Substrate and Conc. | Assay Buffer and Solvent Content | Volumes AP/BL | Preincubationa/Assay time | Plate shaking | Positive control and Conc. |
---|---|---|---|---|---|---|---|---|---|---|---|---|
cells/well | days | nM | µl | min | rpm | µM | ||||||
07 | Netherlands Cancer Institute p. 13-33 | DMEM, 10% FBS, 1X NEAA, 1X l-glutamine, 1X Pen-Strep | 214,000 | 3 | BD Falcon no. 351181 24-well, PET, 1.0 µm | TEER >300 Ωcm2 | 3H-Digoxin 39 | HBSS pH 7.4 +25 mM HEPES 1% DMSO | 200/700 | 30/90 | 90 | GF120918 2 |
04 | Optivia p. 5-30 | DMEM, 10% FBS, 1X Pen-Strep | 150,000 | 3 | Millipore no. PSRP010R5 24-well, PET, 1.0 µm | TEER >90 Ωcm2 | 3H-Digoxin 100 | HBSS pH 7.4 0.5% DMSO | 250/500 | 30/120 | 60 | Verapamil 100 |
06 | National Cancer Institute p. X - 20 | DMEM, 10% FBS + 1X l-glutamine, 1X pen-strep, Fungizone | 175,000 | 7 | Corning no. 3396 24-well, PC, 0.4 µm | Lucifer Yellow: <5% transport | 3H-Digoxin 100 | HBSS pH 7.4 +10 mM HEPES 1% DMSO | 100/600 | 30/60 | No | Verapamil 250 |
05 | National Cancer Institute p. 29-36 | DMEM, 10% FBS | 52,000 | 4 | Millipore no. PSRP010R5 24-well, PET, 1.0 µm | Lucifer Yellow: <0.5 × 10−6 cm/s | Digoxin 5000 | HBSS pH 7.4 +10 mM HEPES 1% DMSO | 400/1000 | 0/60 | No | GF120918 2 |
01/02 | Netherlands Cancer Institute p. 36 | DMEM + glutamax, 10% FBS, 50 IU/ml penicillin, 50 µg/ml streptomycin | 160,000 | 3–4 | BD Falcon no. 351181 24-well, PET, 1.0 µm | Lucifer Yellow: <5 × 10−6 cm/s | 3H-Digoxin 120 | DMEM w/o pyruvate + l-glutamine + 25 mM HEPES + pyridoxine-HCL 1% DMSO | 450/1300 | 30/90 | ∼300 | GF120918 2 |
03 | Netherlands Cancer Institute p. 10 - 20 | DMEM + glutamax, 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin | 450,000 | 3 | Corning no. 3401 12-well, PC, 0.4 µm | TEER >150 Ωcm2 Mannitol: <2.0 × 10−6 cm/s | 3H-Digoxin 50 | HBSS pH 7.4 +25 mM HEPES <1% DMSO | 500/1800 | 15–30/120 | 60 | Ketoconazole 100 |
For abbreviations, see Table 1.
↵a Preincubation in presence of inhibitor.