Computational protocol: Patient-derived hepatitis C virus inhibits CD4+ but not CD8+ T lymphocyte proliferation in primary T cells

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Protocol publication

[…] To quantify proliferation of lymphocytes exposed to HCV or NHP, cells were stained with carboxyfluorescein succinimidyl ester (CFSE), as previously reported []. Briefly, ~2 × 107cells were incubated with CFSE (Molecular Probes, Eugene, Oregon, USA) at the pre-tested nontoxic concentration of 1 mM for 10 min at 37 °C and washed twice with 10 mL of 5 % fetal calf serum (FCS; GIBCO-Invitrogen Corporation, Auckland, New Zealand). To carry out triplicate infection with each of the HCV inocula tested, NHP or NP control, CFSE-labelled lymphocytes were suspended at 1 × 106 cells in one mL of medium containing 10 % FCS in 6-well plates. As an additional control, CFSE-labelled cells in medium alone were included. Cell proliferation was measured in triplicate wells before stimulation with PHA and at 0, 1, 4, 7 and 10 d.p.i., unless otherwise stated. The data were analyzed with Cellquest Pro (Becton Dickinson, San Jose, California, USA) or ModFit LT (Verity Software House, Topsham, Main, USA) software. Using forward versus side scatter, lymphocytes (gate R1) were separated from debris. Proliferation was presented as the mean percentage of CFSE-low cells with standard error of mean (SEM) from triplicate cultures using percentage of CFSE-low cells from cultures maintained in medium alone as the baseline. In the proliferation experiments, CFSE-labelled lymphoid cells were additionally stained with a cocktail of APC-anti-CD3, PerCP-anti-CD4 and PE-anti-CD8 mAbs, as outlined above. Using forward versus side scatter, lymphocytes were separated from debris, gated on CD3+ T cells, and sub-gated on CD4+ or CD8+ T cells to determine the proliferation of individual T cell subsets. Mitogen non-stimulated controls were routinely included, as described above. Dilution of CFSE fluorescence in the cells was measured using the proliferation wizard module of ModFit LT software revealing daughter cell generations. Proliferation index (P.I.) was determined using non-stimulated cells to define the parent generation. […]

Pipeline specifications

Software tools ModFit LT, Wizard
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Hepatitis C, Hepatitis C, Chronic