NFAT Luciferase Reporter-RAW264.7 Cell Line

Product Name

NFAT Luciferase Reporter-RAW264.7 Cell Line

See all NFAT products

SKU/Catalog Number

RC1011

Size

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

Description

The NFAT Luciferase Reporter cell line is a stably transfected RAW264.7 cell line which expresses Renilla luciferase reporter gene under the transcriptional control of the Nuclear Factor of Activated T-cells (NFAT) response element, so that the cell line is designed to measure the transcriptional activity of NFAT. NFAT is a transcription factor originally found in activated T lymphocytes, and is now known to regulate not only T cell activation and differentiation but also the function of other immune cells including dendritic cells, B cells and megakaryocytes. The NFAT induction by calcium ionophore A23187 is shown in Figure 1. 

Contents

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

Gene Name

NFAT

Storage & Handling

Immediately upon receipt, store in liquid nitrogen. (Ship on dry ice.)

Cite This Product

NFAT Luciferase Reporter-RAW264.7 Cell Line (Boster Biological Technology, Pleasanton CA, USA, Catalog # RC1011)

Assay Dilutions Recommendation

The recommendations below provide a starting point for assay optimization. Actual working concentration varies and should be decided by the user.

Application:

Monitor the NFAT signaling pathway activity.Screen for activators or inhibitors of the NFAT signaling pathway. 

Culture conditions:

Cells should be grown at 37°C with 5% CO2 using DMEM medium supplemented with 10% FBS and 1% Pen/Strep, plus 3 µg/ml of Puromycin.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 1~2 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.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. 

Functional validation:

A. Response of NFAT RAW264.7 cells to calcium ionophore A23187. 1. Harvest NFAT RAW264.7 cells and seed cells into a white solid-bottom 96-well microplate in 100 µl of growth medium at 8.5 x 10^4 cells/well.  2. Incubate cells at 37°C in a CO2 incubator for overnight. 3. The next day, stimulate cells with different concentrations of calcium ionophore A23187.  4. Incubate at 37°C in a CO2 incubator for 6-16 hours. 5. Add 50 µl of  luciferase assay reagent  per well.  6. Incubate at room temperature for 1-5 minutes and measure luminescence using a microplate luminometer.   

Validation Images & Assay Conditions

Gene/Protein Information For NFAT (Source: Uniprot.Org, NCBI)

*if product is indicated to react with multiple species, protein info is based on the gene entry specified above in "species".

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