DESCRIPTION

• App Id: Functional Assay
• Product Content: Each vial contains 2 ~ 3 x 10^6 cells in 1 ml of 90% FBS + 10% DMSO.
• Application Note: Application: Monitor the IL-6 induction activity. Screen for activators or inhibitors of the IL-6 signaling pathway. Culture conditions: Cells should be grown at 37°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 3 µg/ml of Puromycin (Note: Puromycin 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°C water-bath, transfer to a tube containing 10 ml of growth medium without Puromycin, spin down cells, resuspend cells in pre-warmed growth medium without Puromycin, transfer resuspended cells to T25 flask and culture in 37°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 between 80~90% adherent, remove growth medium and passage the cells through trypsinization and centrifugation. At first passage, switch to growth medium containing Puromycin. Note: NIH 3T3 cells should be split before they reach 90% confluence; otherwise, they become self-lifted and aggregate irrevisibly. Precoating the cell plates with 0.1% gelatin may prevent NIH 3T3 cells from self-lifting. During cell trypsinization, cells covered enough with trypsin-EDTA solution should be stayed at 37°C for 10 min without agitation. 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. Functional validation: A. Response of IL-6 Leeporter™ – NIH 3T3 cells to lipopolysaccharide (LPS). 1. Harvest IL-6 Leeporter™ – NIH 3T3 cells and seed cells into a white solid-bottom 96-well microplate in 100 µl of growth medium at 5 x 104 cells/well. 2. Incubate cells at 37°C in a CO2 incubator for 4-6 hours. 3. Stimulate cells with various concentrations of LPS. 4. Incubate at 37°C in a CO2 incubator for 16 hours. 5. Equilibrate the plate to room temperature for 10 minutes. 6. Add 50 µl of luciferase assay reagent (Abeomics, Cat #17-1101; 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. B. Response of IL-6 Leeporter™ – NIH 3T3 cells to IL-6. 1. Harvest IL-6 Leeporter™ – NIH 3T3 cells and seed cells into a white solid-bottom 96-well microplate in 100 µl of growth medium at 5 x 104 cells/well. 2. Incubate cells at 37°C in a CO2 incubator for 4-6 hours. 3. Stimulate cells with various concentrations of IL-6. 4. Incubate at 37°C in a CO2 incubator for 16 hours. 5. Equilibrate the plate to room temperature for 10 minutes. 6. Add 50 µl of luciferase assay reagent (Abeomics, Cat #17-1101) per well. 7. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer.
• Storage Condition: Immediately upon receipt, store in liquid nitrogen.
• Shipping Condition: Dry ice