TLR5/NF-kB Luciferase Reporter-HEK293 Cell Line

Product Name

TLR5/NF-kB Luciferase Reporter-HEK293 Cell Line

See all TLR5/NF-kB products

SKU/Catalog Number

RC1039

Size

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

Description

The TLR5/NF-kB Luciferase Reporter cell line is a stably transfected HEK 293 cell line which expresses full-length human Toll-like receptor 5 (TLR5) and Renilla luciferase reporter gene under the transcriptional control of the NF-kB response element. TLR5 is one of the key innate immune receptors. Functional activity of the cell line has been validated by TLR5 ligand assay, in which upon activation by Flagellin, TLR5 quickly initiates downstream signaling pathway and mediates nuclear translocation of NF-kB (Figure 1). 

Contents

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

Gene Name

TLR5/NF-kB

Storage & Handling

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

Cite This Product

TLR5/NF-kB Luciferase Reporter-HEK293 Cell Line (Boster Biological Technology, Pleasanton CA, USA, Catalog # RC1039)

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 TLR5 signaling pathway activity.Screen for activators or inhibitors of the TLR5 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 2 µg/ml of Puromycin and 5 µg/ml of Blasticidin. 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 and Blasticidin, spin down cells, resuspend cells in pre-warmed growth medium without Puromycin and Blasticidin, transfer resuspended cells to T25 flask and culture in 37°C-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 and Blasticidin. 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 TLR5/NF-kB – HEK293 cells to Flagellin. 1. Harvest TLR5/NF-kB – HEK293 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 37°C in a CO2 incubator for overnight. 3. The next day, stimulate cells with various concentrations of Flagellin. 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 TLR5/NF-kB (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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