TET-OFF Leeporter™ GFP Reporter-HEK293 Cell Line

Fig-1: GFP suppression in TET-OFF Leeporter™ GFP reporter-HEK293 cells was analyzed by fluorescence microscopy. Doxycycline was treated at different concentrations as noted.

TET-OFF Leeporter™ GFP Reporter-HEK293 Cell Line

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Fig-1: GFP suppression in TET-OFF Leeporter™ GFP reporter-HEK293 cells was analyzed by fluorescence microscopy. Doxycycline was treated at different concentrations as noted.

Fig-2: GFP suppression in TET-OFF Leeporter™ GFP reporter-HEK293 cells was analyzed by fluorescence microscopy. Doxycycline was treated at different concentrations as noted. Vehicle control (Black line, empty); Doxycycline treatment (Green line, grey-filled).

Product code: 14-704ACL

Application :	Functional Assay

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Shipping Info:

Order now and get it on Friday November 22, 2024

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Download TDS / Manual MSDS

Amount :	1 Vial
Content :	Each vial contains 2 ~ 3 x 10^6 cells in 1 ml of 90% FBS + 10% DMSO.
Storage condition :	Immediately upon receipt, store in liquid nitrogen.

The TET-OFF Leeporter™ GFP Reporter cell line is a stably transfected HEK293 cell line, which expresses enhanced green fluorescent protein (eGFP) reporter gene under the control of the Tet-Off regulatory mechanism. So the cell line can express GFP only when doxycycline is removed from the cell culture medium, and the GFP expression can be turned off by adding doxycycline to the cell culture medium. The cell line is designed to analyze by fluorescence microscopy and flow cytometry. The GFP expression patterns in the presence or absence of doxycycline are shown in Figures 1 and 2.

Application:

Monitor the tetracycline (doxycycline)-controlled GFP expression.
Tetracycline activity.

Culture conditions:

Cells should be grown at 37^oC 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^oC 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^oC-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 TET-OFF Leeporter™ GFP reporter – HEK293 cells to Doxycycline.

1. Harvest TET-OFF Leeporter™ GFP reporter – HEK293 cells and seed cells into a tissue culture plate (e.g. 96-well plate in 100 ul of growth medium at 5 x 10^4 cells/well, 24-well plate in 500 ul of growth medium at 2.5 x 10^5 cells/well, 12-well plate in 1 ml of growth medium at 5 x 10^5 cells/well, or 6-well plate in 2 ml of growh medium at 1 x 10^6 cells/well.

2. Incubate cells at 37^oC in a CO₂ incubator for overnight.

3. The next day, stimulate cells with different concentrations of doxycycline.

4. Incubate at 37^oC in a CO₂ incubator for 16-24 hours.

5.Analyze cells through fluorescence microscopy or flow cytometry.

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 ([email protected]).

For Research Use Only. Not for use in diagnostic/therapeutics procedures.

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