MDA5/NF-kBT Cell Line

Immediately upon receipt, store in liquid nitrogen.

Each vial contains 2 ~ 3 x 10^6 cells in 1 ml of 90% FBS + 10% DMSO.

Cells should be grown at 37 degree C with 5% CO₂ using DMEM medium (w/ L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate) supplemented with 10% heat-inactivated FBS and 1% Pen/Strep, plus 2 ug/ml of Puromycin and 5 ug/ml Blasticidin (Note: Puromycin and Blasticidin can be omitted during the reporter cell assays).

It is recommended to quickly thaw the frozen cells upon receipt or from liquid nitrogen in a 37 degree C water-bath, transfer to atube containing 10 ml of growth medium without Puromycin and Blasticidin, spin down cells, resuspend cells in prewarmed growth medium without Puromycin and Blasticidin, transfer resuspended cells to T25 flask and culture in 37 degree C CO₂ incubator.

Leave the T25 flask in the incubator for 1~3 days without disturbing or changing the medium until cells completely recover viability and become adherent. Once cells are over 90% adherent, remove growth medium and passage the cells through trypsinization and centrifugation. At first passage, switch to growth medium containing Puromycin and Blasticidin. Cells should be split before they reach complete confluence.

To passage the cells, detach cells from culture vessel with Trypsin/EDTA, add complete growth medium and transfer to a tube, spin down cells, resuspend cells and seed appropriate aliquots of cells suspension into new culture vessels. Subcultivation ration = 1:10 to 1:20 weekly. To achieve satisfactory results, cells should not be passaged over 16 times.

A. Response of MDA5 Leeporter™ – HEK293T cells to Poly(I:C).

1. Harvest MDA5 Leeporter™ – HEK293T cells and seed cells into a white solid-bottom 96-well microplate in 100 ul of growth medium at 5 x 10⁴ cells/well.
2. Incubate cells at 37 degree C in a CO₂ incubator for overnight.
3. The next day, stimulate cells with various concentrations of the poly(I:C) packed with lipofectamine 2000, which was prepared as follows:
i) A 50 ul of 10 mg/ml poly(I:C) (= 500 ug total) is preincubated in 50 ul Opti-MEM (Life Technologies) for 5 min.
ii) Similarly, a 20 ul Lipofectamine 2000 (Life Technologies) is preincubated in 80 ul Opti-MEM for 5 min.
iii) After 5 min, they are combined together as a total volume of 200 ul and further incubated for 20 min at room temperature.
iv) The poly(I:C) packed with Lipofectamine 2000 (A 200 ul total at 2.5 mg/ml) is then used to stimulate cells.
4. Incubate at 37 degree C in a CO₂ incubator for 16 hours.
5. Equilibrate the plate to room temperature for 10 minutes.
6. Add 50 ul of luciferase assay reagent (Cat # MBS668917; Refer to the reagent datasheet for the detailed luciferase assay protocol) per well.
7. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer.