Cd3 pacific blue clone sp34 2

Liver cells were freshly isolated from liver tissue as described above. Two million liver cells were pelleted by centrifugation and resuspended in 100 uL of pHrodo E. coli bioparticles (Life Technologies, P35361) prepared at 1 mg/mL in PBS + 2% FBS. Cells were incubated with E. coli bioparticles for 2 hours at 37°C, 5% CO₂. A negative control was conducted using 10 uM cytochalasin D. Cells were washed once with PBS + 2% FBS and then stained with CD3-Pacific Blue clone SP34-2 (BD Biosciences), CD4-BV650 clone OKT4 (Biolegend), CD8-APC-H7 clone SK1 (BD Biosciences), CD45-APC clone MB4-6D6 (Miltenyi), CD14-BV785 clone M5E2 (Biolegend), CD68-FITC clone Y1/B2A (eBioscience), and Live/Dead Aqua Fixable Dead Stain (Life Technologies) for 20 minutes at room temperature. After a wash in PBS + 2% FBS, cells were resuspended in PBS + 2% FBS and acquired on a BD LSRII flow cytometer with phagocytosed E. coli bioparticles detected on the PE filter. Data were analyzed using FlowJo software (version 1.1.0-SNAPSHOT).

The cytokine production profile of HCV-specific T-cell lines was determined by intracellular cytokine staining (ICS) after restimulation with NS3-peptides. For T-cell epitope mapping 0.2×10⁶ expanded cells were either restimulated with NS3-peptide-pool (1 µg/ml/peptide) or without NS3-peptide, in medium containing co-stimulatory αCD28 and αCD49d molecules (2 µg each, BD-Biosciences) and 5% FCS (MP-Biomedicals). After 2 hrs, Brefeldin A (Golgiplug 1∶1000, BD-Biosciences) was added and 16 hours later the surface markers were stained with a panel of fluorochrome labeled antibodies, containing CD3-PacificBlue (clone SP-34-2, (BD-Pharmingen), CD14-PE-TexasRed (clone RMO52, BeckmanCoulter), CD20-PE-TexasRed (clone B9E9, BeckmanCoulter), CD4-PE-Cy7 (clone SK3, BD-Pharmingen) and CD8-APC-H7 (clone SK1, BD-Pharmingen). Stained cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences), followed by staining of accumulated intracellular cytokines with IFNγ-APC (clone B27, BD Pharmingen), IL-2-PE (clone MQ1-17H12, BD Pharmingen) and TNFα-FITC (clone MAb11, BD Pharmingen). Cell staining was analyzed on a FACSAria (BD Bioscience) and DIVA software Version 6.1.1.
To evaluate antigen sensitivity, T-cell lines were restimulated with increasing concentrations of individual peptide ranging from 0.05 to 10 µg/ml before intracellular cytokine production was measured.