Induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice

One-step growth curves were then determined in monocytes harvested from the peripheral blood of A/J, BALB/c and C3H/St mice. For these experiments mice were killed, and PBM were isolated asceptically over Ficoll-Hypaque. Monocytes were separated from lymphocytes by adherence to plastic for 48 h and were >92% esterase positive (38) . Monocytes at 3 × 105 were seeded in 24-well cluster plates (Costar Data Fzc. 1. One-step growth curve of MHV-3 in 2 X 10 7 | 7 CLI cells. Cells were infected in suspension with MHV-3 at an MOI of 0.1 (29) . After virus adsorption for 30 rain at 37°C, the cells were collected by low-speed centrifugation and resuspended in media containing 2% FCS, and replicate cultures were seeded at 1 × 10 e cells per 12 X 35-ram culture dish. At appropriate intervals, cultures were frozen, and the cells were harvested by freeze-thawing, scraped with a rubber policeman, and disrupted by sonication. The homogenates were then clarified by centrifugation, and virus titer was determined by plaque assay at serial 10-fold dilutions. Each data point represents the mean of duplicate cultures, and each line represents individual experiments.