The STAT1 reporter cell line is a stably transfected HeLa cell line which expresses Renilla luciferase reporter gene under the transcriptional control of the interferon (IFN) gamma activation sequence-based STAT1 response element, so that the cell line is designed to measure the transcriptional activity of STAT1. As a transcription factor, Signal Transducer and Activator of Transcription 1 (STAT1) is activated through phosphorylation at tyrosine 701 in response to various cytokines and growth factors such as IFN-alpha, IFN-gamma, IL-6, EGF and PDGF. The phosphorylated STAT1 forms homodimers or heterodimers with STAT3, and the dimers translocate to nucleus in which DNA binding/promoter induction occurs. The STAT1 induction by IFN-gamma is shown in Figure 1.
Application:
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Monitor the STAT1 signaling pathway activity.
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Screen for activators or inhibitors of the STAT1 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 1~3 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. 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.
Functional validation:
A. Response of STAT1 Leeporter™ – HeLa cells to IFN-gamma.
1. Harvest STAT1 Leeporter™ – HeLa cells and seed cells into a white solid-bottom 96-well microplate in 100 µl of growth medium at 5 x 10^4 cells/well.
2. Incubate cells at 37oC in a CO2 incubator for 6-16 hours.
3. The next day, stimulate cells with different concentrations of human IFN-gamma.
4. Incubate at 37oC 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.
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 use if applicable. Please contact Abeomics (info@abeomics.com).
