The MP preparations were characterized by way of fluorescence-activated cell sorting (FACS) with an LSR2 sorter (Becton Dickinson, San Jose, CA) and cytometric data was analyzed with FlowJo 8.8.6 software (Tree Star, Inc., Ashland, Oregon). MP particles were gated on forward and sidescatter acquired from runs including 500 standard beads (Becton Dickinson, San Jose, CA). The number of CD3 (CD11a, CD14, CD147) and AnnexinV (eBioscience, San Diego, CA; GeneTex Inc., Irvine, CA for CD147) double-positive events were calculated relative to the number of beads added to the samples. To avoid unspecific antibody binding, Fc receptors on MPs and target cells were blocked with PD0325901 supplier FcR Blocking Reagent

(Miltenyi Biotec). Antibody solutions were centrifuged prior to FACS to avoid artifacts due to aggregation. Human peripheral blood was collected in citrate-containing tubes (BD Vacutainer, Buff. Na. Citrate [9NC]; BD, Franklin Lakes, NJ) from patients and healthy controls (protocol approved by the Beth Israel Deaconess Medical Center, approval no. 2004-P-000318).

MPs were isolated by way of differential centrifugation, and S100-MPs were characterized by way of FACS using staining for Annexin PARP phosphorylation V, CD3, CD4, CD8, CD14, CD15, CD41, and CD25 (eBioscience) as detailed above. Levels of T cell MPs were correlated with liver histology as detailed in the Supporting Materials and Methods. HSCs (200 × 103/well) were seeded into six-well culture plates

and serum-starved for 24 hours, followed by incubation with 1 × 103 or 50 × 103 S10-MPs or S100-MPs for 24 hours, and RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA). ST (0.04 μM/mL) or plain medium served as controls. One microgram of RNA was reverse-transcribed using random primers and Superscript RNase H-reverse transcriptase (Invitrogen). Primers and probes are listed in Supporting Table 2. Relative transcript levels were quantified on a LightCycler 1.5 (Roche, Mannheim, Germany) using the TaqMan methodology. MP membranes were labeled with the PKH26 lipid dye (Sigma-Aldrich, St. Louis, MO). Labeled S10-MPs and S100-MPs were coincubated with LX-2 cells for 0-1, 30, and 60 minutes, washed extensively, and fixed with 2% paraformaldehyde for 15 minutes at room O-methylated flavonoid temperature. HSCs (200 × 103/well) were seeded into six-well cell culture plates (BD Labware, Franklin Lakes, NJ) for 12 hours, serum-starved for 24 hours, followed by incubation with 100 × 103 S100-MPs for 1 minute up to 24 hours. Cells were then washed with phosphate-buffered saline and collected using trypsin/ethylene diamine tetraacetic acid (Cellgrow, Manassas, VA), and single-cell suspensions stained with anti–CD3-APC were followed by FACS analysis. Tumor necrosis factor α (TNFα) (PeproTech) was added to HSC cultures, and ICAM-1 expression assessed after 2, 4, and 24 hours by FACS using anti–ICAM-1 PE (eBioscience, San Diego, CA).