Infection of bovine PBMC. PBMC were isolated from bovine (Holstein Breed) whole blood on Ficoll density gradient and cultured with RPMIc containing RPMI 1640 with Glutamax, 25 mM Hepes (Gibco-BRL) supplemented with 10 % FCS, 1 % sodium pyruvate, 1 % non essential amino acids, 1 % β-mercapto-ethanol, 100 units/ml penicillin, 100 μg/ml streptomycin. When indicated, cells were activated 4 h before infection, using 125 ng/ml of phorbol myristate acetate and 50 ng/ml of ionomycine. To determine cell viability, propidium iodide (BD Biosciences Pharmingen) was added at 1 μg/ml just before acquisition. Acquisition was performed using a FACScalibur (Becton Dickinson). Dead cells and debris were excluded by appropriate gating and 30000 events were collected. Analysis was performed using CellQuestPro and Flowjo Software. A. Cells were infected at m.o.i. of 1 (T1-Serp2-GFP or SG33-GFP) and collected 16 h p.i.. Results shown are representative of three experiments. B. Cells were infected at m.o.i. of 1 (T1-Serp2-GFP or SG33-GFP), collected 16 h p.i and analyzed for GFP expression by flow cytometry. The percentages indicated represent an average of at least 3 independent experiments. Errors bars correspond to the standard error of the mean. C. PBMC were inoculated with T1 or SG33 (m.o.i. = 1), adsorption occurring 90 min at 4°C. Cells were washed twice and cultured. Virus productions at 0 h or 72 h post-infection were determined by serial dilution-titration on RK13 cells. The experiment was repeated three times. Error bars correspond to the standard error of the mean.
Pignolet et al. Virology Journal 2007 4:94 doi:10.1186/1743-422X-4-94