Technical Reference #1936

1936.

Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in Freshly Isolated Rat Hepatocytes Daniel A.J. Bow; Jennifer L. Perry; David S. Miller; John B. Pritchard; and Kim L. R. Brouwer, University of North Carolina at Chapel Hill, Drug Metabolism and Disposition, 36(1936), (2008)
Link To Paper

Materials & Methods:
Isolation and Culture of Rat Hepatocytes. Hepatocytes were isolated from male wild-type or TR Wistar rats using a collagenase perfusion method as described previously (Liu et al. 1999). Hepatocyte viability was 85% as determined by trypan blue exclusion. Immediately after isolation suspended hepatocytes were centrifuged for 5 min at 50g and then resuspended in phosphate-buffered saline (PBS) at 106 cells/ml. Cells were allowed to attach to 35-mm glass-bottomed culture dishes (glass coverslip; 14-mm diameter 0.16–0.19-mm thickness) (MatTek Corporation Ashland MA) previously coated with rat tail collagen solution (75 l of type I collagen; 1.5 mg/ml pH 7.4) at room temperature (RT) for 30 to 60 min. For the purpose of this study cells allowed to attach to dishes at RT were described as “freshly isolated.” For the 1-h culture period hepatocytes (0.75–1.5 106) were seeded in Dulbecco’s modified Eagle’s medium (supplemented with 5% fetal bovine serum nonessential amino-acids L-glutamine penicillin/streptomycin and 1 M dexamethasone) on the collagen-coated 35-mm glass-bottomed culture dishes and cultured in a humidified incubator with 95% air/5% CO2.

Microscopic Technique
Laser scanning confocal microscope

Cell Type(s)
Hepatocytes