6% were CD11c+. PHHs shipped in plates were incubated with InVitroGRO HI medium (Celsis)

for 4 hours at 37°C (5% CO2), followed by polyinosinic/polycytidylic acid (polyI:C) transfection or HCV infection, as described below. PHHs shipped in suspension were centrifuged at 50×g for 5 minutes, incubated in InVitroGRO CP medium (Celsis) in 12- (7 × 105/well) or 24-well (3.5 × 105/well) plates overnight, and transfected with 5 μg of polyI:C (InvivoGen, San Diego, CA) and 6.4 μL of Lipofectamine 2000 in Opti-MEM medium (Invitrogen), or infected with HCV (Japanese fulminant hepatitis type I [JFH-1] strain)22 at a multiplicity of infection (MOI) of 0.4-2.7. After 3-6 hours, culture medium was replaced with InVitroGRO HI including Torpedo antibiotic mix (Celsis). In some experiments, PHHs were incubated 30 minutes before HCV infection PD0325901 mw with neutralizing antibodies (Abs) against type I IFNs (10 μg/mL each of anti-IFN-α [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-β [R&D Systems, Minneapolis, MN]) or type III IFNs (10 μg/mL each of polyclonal anti-IL-29, polyclonal anti-IL-28A, and anti-IL-29/IL-28B [clone 247801;, http://www.selleckchem.com/EGFR(HER).html R&D Systems]). Total RNA was isolated

from snap-frozen, mechanically homogenized liver biopsies or from PHH using the RNeasy Mini Kit (Qiagen, Valencia, CA) with on-column DNase digestion. A complementary DNA (cDNA) equivalent to 20-80 ng of total RNA, generated with the MonsterScript 1st-Strand cDNA Synthesis Kit (EPICENTRE Biotechnologies, Madison, WI), was used to determine IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), myxovirus resistance 1 (MX1), chemokine (C-X-C motif) ligand (CXCL)10, CXCL11, IFN-α2, IFN-β, IL-29, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and proteasome subunit, beta type, 4 (PSMB4) 上海皓元医药股份有限公司 messenger RNA (mRNA) levels with predesigned human TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). Because IL28A and IL28B

share 98% of their nucleotide sequence, their mRNA levels were quantitated with shared forward primer 5′-TGAGGCAGCTGCAGGTGA-3′, reverse primer 5′-CTCCAGAACCTTCAGCGTCAG-3′, and probe 5′-FAM-TGGCTTTGGAGGCTGA-MGB-3′ designed with Primer Express (Applied Biosystems). The specific mRNA amount was quantitated using comparative cycle threshold values and 1-107 copies/well standard curves, and normalized to mean GAPDH and PSMB4 mRNA levels. Relative mRNA levels represent fold-increase over pre-infection or pre-transfection samples. For HCV RNA quantitation, we used TaqMan EZ RTPCR Core Reagents (Applied Biosystems) and forward primer 5′-CGGGAGAGCCATAGTGG-3′, reverse primer 5′AGTACCACAAGGCCTTTCG-3′, and probe 5′-FAM-CTGCGGAACCGGTGAGTACAC -TAMRA-3′ (Sigma-Aldrich). RT-PCR conditions are 2 minutes at 50°C, 30 minutes at 60°C, 5 minutes at 95°C, followed by 50 cycles at 95°C for 20 seconds and at 60°C for 1 minute. HCV RNA levels were normalized to microgram of total input RNA.