NFAT Reporter – RAW264.7 Cell Line

Product code: 14-111ACL

Application : Functional Assay

Shipping Info:

2000 (throughout India)
Order now and get it on next 3-4 working days* (Major Cities)

Size
Price
1 Vial
70,000.00 


Bulk Order


Shipping Info:

2000 (throughout India)

Order now and get it on next 3-4 working days* (Major Cities)


Amount : 1 Vial

The NFAT 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. 

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.

Application:

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

Culture conditions:


Cells should be grown at 37oC 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 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 2~4 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 Leeporter™ – RAW264.7 cells to calcium ionophore A23187.
 
1. Harvest NFAT Leeporter™ – 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 37oC in a CO2 incubator for overnight.
 
3. The next day, stimulate cells with different concentrations of calcium ionophore A23187. 
 
4. Incubate at 37oC in a CO2 incubator for 6-16 hours.
 
5. Add 30-50 µl of  luciferase assay reagent  per well. 
 
6. Incubate at room temperature for 1-5 minutes and measure luminescence using a microplate luminometer.
 


NFAT Reporter – RAW264.7 Cell Line

Product code: 14-111ACL
mg

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