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ITS 1X is a sterile, protein-free, liquid culture supplement for cell and tissue culture applications. It provides a defined combination of insulin, transferrin, and selenium to support cell growth and performance.

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2 protocols using its 1x

1

Infection Assay with Immortalized Bronchial Epithelial Cells

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Human bronchial epithelial cells (AALEB), immortalised through specific transfection with the simian virus 40 early region and the telomerase catalytic subunit hTERT, were used in infection assays and have previously been described (Lundberg et al., 2002 (link)). AALEB cells were grown in Bronchial Epithelial Growth Medium (BEBM plus SingleQuots of Growth Supplements) (Lonza). AALEB cells were grown to 80% confluency in 24 well plates coated with collagen. AALEB cells were serum starved for 24 h in BEBM supplemented with ITS 1X (insulin, transferrin, selenium, Thermo Fisher Scientific) and 0.02% BSA (Sigma-Aldrich) and infected with a clinically relevant RSV-A (Memphis 37) strain, originally isolated by DeVincenzo et al. (DeVincenzo et al., 2010 (link)). Cells were infected with either a low (multiplicity of infection (MOI) of 0.08) or high (MOI of 0.4) dose of RSV diluted in DMEM (4 mM l-glutamine). RSV was preincubated with varying concentrations of nhSP-A, rfhSP-A, dimeric rfhSP-A or BSA diluted in DMEM (4 mM l-glutamine). Cells were infected for 2 h, after which they were washed and left for 24 h in BEGM media without serum but with recommended supplements.
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2

Apoptosis Detection in Treated Cells

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Isolated cells were expanded to Passage 1 (P1) in monolayer cultures. P1 cells were then seeded at 20,000 or 10,000 cells per well in eight-well chamber slides (Nunc Lab-Tek II Chamber Slide System) and 96- well flat clear bottom black microplates (Corning, NY) respectively. Cells were serum-starved in DMEM with ITS (1X) (Thermo Fisher, Waltham, MA) for 2 hr prior to treatment with 5 μM RG-7112 (Selleck Chemicals, TX), 100 μM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada) or vehicle (DMSO (0.01%, (Sigma-Aldrich, Oakville, ON, Canada) for 6 hr. Immunocytochemistry was performed as previously described (Cherif et al., 2019 (link)). Apoptosis was detected using a commercial kit (ab176749, Abcam, Cambridge, MA) according to the manufacturer’s instructions. Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera (Olympus, Tokyo, Japan).
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