Viral stock production was done by vRNA transfection using Lipofectamine RNAiMax® (Thermo Fisher Scientific, Waltham, MA). Transfection reagent (1μg/ml) was added to NCI-H1299 cells seeded in a 12-well tissue culture plate at 1 × 105 cells/well. After 72 h post-transfection, supernatants were collected, centrifuged at 2000 × g for 5 minutes, and filtered through a 0.45 μm filter. Filtered supernatant (100 μl) was used to infect a new 12-well plate seeded with NCI-H1299 cells. After 72 h, cells and supernatants were subjected to 3× freeze-thaw cycles, then centrifuged and filtered. Supernatant (1 mL) was then used to infect NCI-H1299 cells grown to 80% confluency in a two-chamber CellSTACK® (Corning, Corning, NY) tissue culture vessel in 250 ml of growth media. After 96 h post-infection, the cells and supernatants were 3× freeze-thawed, centrifuged, filtered, and concentrated using 100K Amicon® Ultra 15 Centrifugal Filter Units (Millipore, Burlington, MA) to a 10 ml final volume which was aliquoted and stored at −80 °C.
100k amicon ultra 15 centrifugal filter units
Manufactured by Merck Group
The Amicon® Ultra 15 Centrifugal Filter Units are a lab equipment product from Merck Group. They are designed for fast and efficient concentration and desalting of protein and other macromolecular samples. The filter units have a molecular weight cutoff of 100,000 Daltons and can be used with a standard laboratory centrifuge.
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Lab products found in correlation
2 protocols using 100k amicon ultra 15 centrifugal filter units
1
Viral Stock Production via Lipofectamine Transfection
2
Viral Stock Production by vRNA Transfection
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Viral stock production was done by vRNA transfection using Lipofectamine RNAiMax ® (Thermo Fisher Scienti c, Waltham, MA). Transfection reagent (1μg/ml) was added to NCI-H1299 cells seeded in a 12well tissue culture plate at 1 x 10 5 cells/well. After 72 h post-transfection, supernatants were collected, centrifuged at 2000xg for 5 minutes, and ltered through a 0.45-micron lter. Filtered supernatant (100 μl) was used to infect a new 12-well plate seeded with NCI-H1299 cells. After 72 h, cells and supernatants were subjected to 3X freeze-thaw cycles, then centrifuged and ltered. Supernatant (1 mL) was then used to infect NCI-H1299 cells grown to 80% con uency in a two-chamber CellSTACK® (Corning, Corning, NY) tissue culture vessel in 250 ml of growth media. After 96 h post-infection, the cells and supernatants were 3X freeze-thawed, centrifuged, ltered, and concentrated using 100K Amicon ® Ultra 15 Centrifugal Filter Units (Millipore, Burlington, MA) to a 10 ml nal volume which was aliquoted and stored at -80 C.
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