2, 3 Woodchuck WCM-260
hepatocyte cell line was established from the liver of a healthy woodchuck and maintained as reported.2, 3 Hepatocytes were isolated from livers of CDI mice (Charles River Laboratories, Wilmington, MA) by way of two-step collagenase microperfusion, as reported.2, 3, 14 Preparations were at selleck screening library least 98% pure on phase-contrast microscopy. Murine splenocytes and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation as described.18 CD4+CD25− T cells were affinity-purified from murine splenocytes using magnetic bead separation (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. In some experiments, splenocytes, PBMCs, and purified T cells were stimulated for 48 hours with 10 μg/mL phytohemagglutinin (PHA) (Sigma-Aldrich, Oakville, Ontario, Canada) in the presence of 10 U/mL human recombinant interleukin-2 (Roche Diagnostics, Pleasanton, CA) prior to labeling with 3H-thymidine for an additional 18 hours. Animal experimental protocols were
approved by the Institutional Presidents’ Committee on Animal Bioethics and Care. Total RNA was extracted from primary hepatocytes using Trizol reagent (Invitrogen, Carlsbad, CA). Potential DNA contamination was removed using a DNase digestion kit (Sigma-Aldrich). RNA (2 μg) was reverse-transcribed to complementary DNA as reported.4 Real-time reverse-transcription polymerase chain reaction (RT-PCR) was established to quantify transcription of the ASGPR-1 major subunit using the gene exon-specific forward Ivacaftor ic50 primer 5′-CAGTGAGTCCTATCCAGA-3′ and reverse Molecular motor primer 5′-AGA GCAATGGCACAGA-3′, the LightCycler Faststart Master SYBR I kit (Roche Diagnostics, Laval, Quebec, Canada), and the Roche LightCycler (Roche Diagnostics). β-Actin–specific primers and amplification conditions have been described.4 WCM-260 hepatocytes and HepG2 cells were grown to confluence (≈6 × 104 cells/well)
in 96-well flat-bottom cell culture plates, and primary fresh mouse hepatocytes were aliquoted at 6 × 104/well in 200-μL volumes. 3H-thymidine–labeled P815 or K562 cells, or activated murine splenocytes, PBMCs, or CD4+ T cells, were added at 4 × 104 cells per well as described.4 Plates were centrifuged for 5 minutes at 45g and incubated at 37°C in a 5% CO2 atmosphere for 18 hours. Well contents were harvested onto glass fiber mats (Perkin Elmer, Wellesley, MA) using a 96-well harvester (Tomtec, Hamden, CT). Counts per minute were measured using a Top10 beta counter (Becton Dickinson, San Diego, CA), and percent lysis was determined as described.4 Inhibition of microtubule-dependent granule release was facilitated using 1 mM colchicine (Sigma-Aldrich).4 Where indicated, target cells were pretreated for 1 hour at 37°C with titrated amounts (0.01, 0.1, 0.5 U/mL) of neuraminidase type VI (Sigma) as described.