To capture single hRSV filamentous virions on glass, hRSV A2 was propagated in HEp-2 cells at an MOI of 0.1. At 4 days post infection, the cell-associated and supernatant fractions were scraped, freeze-thawed and spun through 5 and 0.45 µm pore-size centrifugal filters (EMD Millipore) at 5000 × g and 4 °C for 4 and 1 min, respectively. The fraction between 0.45 and 5 µm in diameter was collected and immobilized onto a poly-l -lysine (Sigma Aldrich)-coated, first-surface mirror or cover glass by adsorption of 500 µl of filtered virus for 2 h at 4 °C. The immobilized virions were fixed using 4% paraformaldehyde and were immunofluorescently stained according to the aforementioned protocol. The antibodies used were anti-RSV F monocolonal (palivizumab, MedImmune, Gaithersburg, MD, USA) and anti-RSV N monoclonal (monoclonal IgG1, ab22501, Abcam). Coverslips were mounted in a mixture of Mowiol and DABCO (VWR)19 (link).
Pore size centrifugal filters
Manufactured by Merck Group
Pore-size centrifugal filters are laboratory equipment used to separate and concentrate molecules, cells, or other particles based on their size. They operate by applying centrifugal force to drive the sample through a membrane with specific pore sizes, allowing the desired components to be retained while the smaller elements pass through.
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2 protocols using pore size centrifugal filters
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Capturing hRSV Filamentous Virions
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Capturing hRSV Filamentous Virions
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To capture single hRSV filamentous virions on glass, hRSV A2 was propagated in HEp-2 cells at an MOI of 0.1. At 4 days post infection, the cell-associated and supernatant fractions were scraped, freeze-thawed and spun through 5 and 0.45 μm pore-size centrifugal filters (EMD Millipore) at 5000 × g and 4 °C for 4 and 1 min, respectively. The fraction between 0.45 and 5 μm in diameter was collected and immobilized onto a poly-l -lysine (Sigma Aldrich)-coated, first-surface mirror or cover glass by adsorption of 500 μl of filtered virus for 2 h at 4 °C. The immobilized virions were fixed using 4% paraformaldehyde and were immunofluorescently stained according to the aforementioned protocol. The antibodies used were anti-RSV F monocolonal (palivizumab, MedImmune, Gaithersburg, MD, USA) and anti-RSV N monoclonal (monoclonal IgG1, ab22501, Abcam). Coverslips were mounted in a mixture of Mowiol and DABCO (VWR)19 (link).
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