Permissiveness of Guinea Pig Alveolar Macrophage Subpopulations to Acute Respiratory Syncytial Virus Infection In Vitro: Preparation of AM Subpopulations

In: Respiratory

20 Jul 2014

Accordingly, we designated the subpopulation of AMs recovered at the interface 20 to 22% metrizamide as “high-density” AMs; cells recovered at the interface 18 to 20% metrizamide were called “intermediate-density” AMs; and those collected on top of the 18% metrizamide gradient were designated as “hypodense” AMs. The purity of AMs recovered from each fraction was 100% in hypodense, 96% in intermediate-density, and 72% in liigh-density cells. In the latter fraction, contaminating cells consisted most of eosinophils (24%). Morphologically, hypodense AMs were the largest cells, had vacuolated cytoplasm, and showed a low nuclear to cytoplasmic ratio; in contrast, liigh-density AMs were smaller and showed a high nuclear to cytoplasmic ratio.
In Vitro Exposure to RSV
The isolated cells were counted and their number adjusted to approximately 10 macrophages (monocytes) per milliliter. Five hundred microliters of each cell suspension were added to each well in six-well culture plates and the cells were allowed to adhere for 60 min at 37°C in 5% C02-containing atmosphere fully buy dexone online. After removing nonadherent cells by washing with MEM, the remaining adherent cells (ie, all of which were macrophages/monocytes, as confirmed by MR-1 antibody staining of designated aliquots) were exposed to RSV at a multiplicity of infection (moi) of 1 at 37°C for 90 min. During this incubation period, the culture plates were manually agitated every 15 min to ensure exposure of cells to the virus. After two washes with MEM to remove nonadsorbed virus, the infected cultures were maintained at 37°C for 24 h before processing for RSV immunocytochemistry, plaque assays, and iminunogold transmission electron microscopy. The moi and duration of culture postinfection were chosen based on our preliminary RSV immunostaining of virus-exposed, unfractionated BAL cells, in which similar percentages of RSV-positive AMs were observed at moi = 1 or 2 (mean ± SD: 26.5 ± 3.1% vs 28.8 ± 4.6%, respectively) at 24 li postinfection, with no further increase by 48 h. Cells were examined for evidence of detachment from plastic 24 h after in vitro exposure to RSV, and AM viability was assessed by trypan blue dye exclusion.

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