Jurkat JE6.1 NF-kB::eGFP hTLR4 (human Toll-like Receptor 4) Cell Line

Contamination with bacterial components is a potential issue with the biologics for research- and therapeutic uses, as they can activate the so-called pattern-recognition receptors in mammalian cells and trigger immune responses, clouding the experimental results or even causing health hazards. Such responses are often mediated by the Toll-like Receptors (TLRs), which recognize bacterial molecular patterns to activate, among other things, cytokine expression through the NF-κB pathway.

The current assessment method for bacterial contamination measures lipopolysaccharide (LPS), the active component of endotoxins. Endotoxin measurement relies on its reaction with the limulus amoebocyte lysate (LAL), derived from the horseshoe crab blood.3 While the method is well established, however, the use of primary lysate obtained from wild animals presents a major sustainability issue.4 Recombinant alternatives to the primary LAL method have recently been gaining acceptance, but all LAL-based methods measure only the endotoxin contamination, and other classes of bacterial components go undetected.

To address these unmet needs, the junket JE6.1 cell line has been engineered to form a group of cell lines, each expressing a specific set of TLRs and harboring an eGFP expression cassette fused to the NF-κB Response Element. The result is that each of these strains expresses eGFP in response to a specific class of bacterial molecules.

SCC630, Jurkat JE6.1 NF-κB::eGFP hTLR4 cell line, expresses recombinant TLR4 and its co-receptors CD14 and MD-2 on its surface and is designed to express eGFP in response to LPS. It is not responsive to other bacterial components, such as the Gram positive- and Gram negative flagellins (ligands for SCC631, the TLR5 cell line), and FSL-1 (ligand for SCC632, the TLR2/TLR6 cell line). It has also been tested negative for PAM3CSK4 (ligand for TLR2/TLR1).

Samples containing high concentration of LPS can be assayed by their serial dilution in the growth medium, and the limit of detection in such experiments is in the range of 0.5 pg/ml (0.005 EU/ml). Samples of low LPS content may be assayed by their direct addition to the growth medium in volumes not exceeding 10% of the medium, and the limit of detection in such reactions is in the range of 5 pg/ml (0.05 EU/ml). Only water and Dubecco′s phosphate-buffered saline (DPBS) have been validated in our hands as the solvents for this purpose, but other vehicles may be appropriate. If components other than LPS are suspected of the resulting eGFP activation, the SCC635 cells (the non-responder cell line) can be used as a negative control.

Source
SCC635, the parental cell line for SCC630, was made by the deletion of the TLR5 gene in the Jurkat JE6.1 cell line harboring the eGFP expression casette fused to the NF-kB Response Element.1,5 SCC635 was retrovirally transduced with the recombinant hTLR4, hCD14, hMD-2 to yield SCC630.

Jurkat JE6.1 T NF-ĸB::eGFP hTLR4 cell line expresses recombinant TLR4 and its co-receptors CD14 and MD-2 and is designed to express eGFP in response to bacterial comtaminants LPS.

Jurkat JE6.1 NF-kB::eGFP hTLR4 cells should be stored in liquid nitrogen. The cells can be cultured for at least 10 passages without significantly affecting cell marker expression and function.

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