Application:
Monitor the JAK/STAT signaling pathway activity.
Screen for activators or inhibitors of the JAK/STAT signaling pathway.
Culture conditions:
Cells should be grown at 37oC 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 37oC 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 37oC-CO2 incubator.
Leave the T25 flask in the incubator for 1~2 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. Cells should be split before they reach complete confluence. Note: RAW264.7 cells may not be detached well by trypsinization only. So you may need to use a cell scraper to harvest the trypsinized cells.
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 ISRE Leeporter™ – RAW264.7 cells to poly(I:C).
1. Plate ISRE Leeporter™ – RAW264.7 cells into a white solid-bottom 96-well microplate in 100 ul of growth medium at 1 x 105 cells/well and incubate cells at 37oC in a CO2 incubator for 4-6 hours.
2. Stimulate cells with different concentrations of poly(I:C) and incubate cells at 37oC in a CO2 incubator for 16 hours. ,
3. Equilibrate the plate to room temperature for 10 minutes.
4. Add 50 µl of luciferase assay reagent (Abeomics, Cat #17-1101; Refer to the reagent datasheet for the detailed luciferase assay protocol) per well.
5. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer.
LIMITED USE RESTRICTIONS:
THIS PRODUCT IS SOLELY FOR IN VITRO RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE.
By use of this product, user agrees to be bound by the terms of this limited use statement.
This product is solely for Internal Research Purposes and not for Commercial Purposes. Commercial Purposes include, but are not limited to (1) use of the cell line in manufacturing; (2) use of the cell line to provide a service, information or data; (3) use of the cell line for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the cell line whether or not such cell lines are resold for use in research. The buyer cannot sell, give or otherwise transfer this product to a third party.
Commercial License Agreement is available for non-research/ non-inhouse/ commercial use if applicable.
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