Louis, MO). Dulbecco’s modified Eagle medium, Liebowitz 15, Fluo-4/acetoxymethyl ester (AM), Cell tracker green 5-chloromethylfluorescein diacetate (CMFDA), the nuclear stain TO-PRO-3, rhodamine-conjugated phalloidin, and Alexa-488 secondary antibodies were obtained from Invitrogen (Eugene, OR). InsP3R1 antibodies from affinity-purified specific rabbit polyclonal U0126 manufacturer antiserum directed against the 19 C-terminal residues of the mouse InsP3R1 were from Affinity Bioreagents (Golden, CO). InsP3R2 antibodies from affinity-purified specific
rabbit polyclonal antiserum directed against the 18 C-terminal residues of the rat InsP3R2 were provided by Richard Wojcikiewicz (SUNY Syracuse, NY). Monoclonal antibodies directed against the N-terminal of InsP3R3 were obtained from Becton Dickinson (Lexington, KY). Bilirubin total reagent was obtained from ClinicQA (San Marcos, CA). Mammalian protein extraction reagent cell lysis buffer was obtained from Pierce (Rockford, IL). Rat GFP-MRP2 expression vector was provided by Dietrich Keppler (German Cancer Research Center, Heidelberg, Germany).27 HepG2 cells were purchased from ATCC (Manassas, VA). All other chemicals AP24534 concentration were of the highest quality commercially available. Isolated mouse hepatocyte couplets were used for single-cell imaging. Cells were
isolated in the Cell Isolation Core of the Yale Liver Center, as described.28, 29 Briefly, mouse livers were perfused with Hanks’
A and then Hanks’ B medium containing 0.05% collagenase (Roche Applied Science, Indianapolis, IN) and 0.8 units of trypsin inhibitor (Sigma) per unit of tryptic activity. Livers were minced and passed through serial nylon mesh filters, and the resultant cells were washed. Isolated hepatocytes were resuspended in Liebowitz 15 medium with 50 units penicillin and 50 mg streptomycin. Cells of were then seeded onto collagen-I-coated coverslips and incubated at 37°C for 2 to 4 hours before use in isolated hepatocyte experiments. For hepatocytes in collagen sandwich culture, cells were incubated for 2 hours at 37°C before being coated with a second layer of collagen-I and used 3 to 5 days after plating.30 For bile flow studies, bile was collected using Intramedic Polyethylene Tubing (Becton Dickinson, Franklin Lakes, NJ) inserted into the gallbladder. Bile was collected in pre-tared tubes, and then flow measurements were normalized by the liver weight and expressed as microliters per gram of liver per minute (μL/g liver/minute). During the entire experiment (1 hour), animals were under anesthesia by inhalation of a mixture of oxygen (0.5 L/minute) and isoflurane (2.5%-3.0%) and their temperature monitored. Immunoblots were performed as described previously.