Author: Suddala, Krishna C.; Lee, Christine C.; Meraner, Paul; Marin, Mariana; Markosyan, Ruben M.; Desai, Tanay M.; Cohen, Fredric S.; Brass, Abraham L.; Melikyan, Gregory B.
Title: Interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes Document date: 2019_1_14
ID: 15wxk8lt_71
Snippet: A549 cells were seeded onto 35 mm collagen-coated glass-bottom Petri dishes (MatTek, MA) one day before imaging and cultured in Fluorobrite DMEM supplemented with 10% FBS, penicillin, streptomycin and L-glutamine. Before imaging, the cells were pre-chilled on ice and washed with ice-cold PBS+/+. A small amount of viral suspension (~1 μL) diluted in 60 μL of cold LCIB was added to the cells and spinoculated at 1550xg at 4˚C for 20 min. After sp.....
Document: A549 cells were seeded onto 35 mm collagen-coated glass-bottom Petri dishes (MatTek, MA) one day before imaging and cultured in Fluorobrite DMEM supplemented with 10% FBS, penicillin, streptomycin and L-glutamine. Before imaging, the cells were pre-chilled on ice and washed with ice-cold PBS+/+. A small amount of viral suspension (~1 μL) diluted in 60 μL of cold LCIB was added to the cells and spinoculated at 1550xg at 4˚C for 20 min. After spinoculation, the cells were washed twice with PBS +/+ to remove any unbound viruses and a small volume (~150 μL) of ice-cold LCIB was added to the cells. Viral entry was initiated by adding 2 mL of pre-warmed (37˚C) LCIB containing 2% FBS, and cells were imaged immediately on DeltaVision microscope equipped with a temperature and humidity-controlled chamber. Every 6-8 sec, at least three Z-stacks spaced by 1.5-2 μm were acquired to cover the thickness of cells using Olympus 40x UPlanFluo 40x/1.3 NA oil objective (Olympus, Japan). The threecolor viral fusion experiments were done with A549 cells expressing IFITM3-imTFP1 or 2M-imTFP1 using a standard CFP/YFP/Cherry filter set (Chroma, VT), while a TRITC/FITC/Cy-5 filter set was used for the three-color lipid mixing experiments with IFITM3-mNeonGreen expressing cells. LCIB in all the experiments was supplemented with 2% FBS.
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