Western Blot: Human TLR4 Stable Cell Line [NBP2-26268] - Analysis of TLR4 expression in the TLR4 stable cell line using an HA antibody (20 ug total protein/lane). Legend. Vect: Vector control stable cell line; TLR4: ...read more
Flow Cytometry: Human TLR4 Stable Cell Line [NBP2-26268] - Analysis of the TLR4 stable cell line. The assay was performed using the NF-kB SEAPorter™ Assay Kit. The Vector control stable cell line and TLR4 stable ...read more
Flow Cytometry: Human TLR4 Stable Cell Line [NBP2-26268] - Flow analysis of TLR4 expression in the TLR4 stable cell line. Cell surface expression of TLR4 in the TLR4 stable cell line was analyzed by flow cytometry ...read more
The TLR4 stable cell line can be used for TLR4 flow cytometric calibration and detection control as well as TLR4-dependent functional assays. TLR4 expression in this stable cell line has been validated by Western blotting (Fig. 1) and flow cytometry (Fig. 2). Functional activity of this stable cell line has been validated by the NF-kB/SEAPorte™ Assay Kit (NBP2-25286, Fig. 3).
Contents: 3~4 x 10^6 cells
Biosafety Level: 2
The TLR4 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 4 (TLR4) with an N-terminal HA tag.
Stable Cell Line
Use in Functional reported in scientific literature (PMID26248657)
Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should be observed. - Use pipette aids to prevent ingestion and keep aerosols down to a minimum. - No eating, drinking or smoking while handling the stable line. - Wash hands after handling the stable line and before leaving the lab. - Decontaminate work surface with disinfectant or 70% ethanol before and after working with cells. - All waste should be considered hazardous. - Dispose of all liquid waste after each experiment and treat with bleach.
Alternate Names for Human TLR4 Stable Cell Line
hTollhomolog of Drosophila toll
toll-like receptor 4
This product is for research use only and is not approved for use in humans or in clinical diagnosis. Reporter Cell Lines are guaranteed for 1 year from date of receipt.
FAQs for TLR4 Reporter Cell Line (NBP2-26268). (Showing 1 - 3 of 3 FAQs).
Have you tested whether NB100-56566/ NB100-56728 can detect TLR4/TLR2 from NBP2-26268/NBP2-26266, respectively? If you tested, could you please inform us about the western blot procedure?
These cell lines are designed for flow cytometry and TLR functional assays. They are not designed for western blot analysis of TLRs. The antibodies that you mentioned do recognize the TLRs in these cells. As shown on the NBP2-26266 data sheet, the TLR2 antibody can be used in flow cytometry to recognize the TLR2 in the NBP2-26266 cell line. As shown on the NBP2-26268 data sheet, the antibody can be used in flow cytometry to recognize the TLR4 in the NBP2-26268 cell line. Please note, in general it is not uncommon that antibodies do not recognize certain recombinant constructs in one or more techniques. Our cell lines were selected for functional activity rather than for recognition by particular TLR antibodies. The construct insert was confirmed by using antibody to HA tag as shown on the data sheets.
The customer stimulated the products TLR4 Stable Cell Line (NBP2-26268) and TLR2 Stable Cell Line (NBP2-26266) with their ligand, and he detected the expression of Defencin by semi-quantitative PCR. Defencin is the response factor downstream of TLR signaling. However the expression level is same to normal HEK cell. When he also used HaCat cell, the expression level of Defencin increased commensurately. He suggests the HaCat cell line seems to be better response than these products. Do you know why this is? Also, TLR2 exists in complex with TLR1 or TLR6 in native condition. TLR4 exists in complex with MD2 and CD14. He noticed whether the TLR in these cell line are not over-expressd, because these cell lines does not over-express their complex element together. He would like to make sure that the expression level of TLR in IMGENEX TLR cell line is higher than native cell line. Additionally he wants to know whether TLR can be detected by using TLR antibody in western blotting generally. Tag antibody can be not used for such analysis, because Tag antibody can not detect native endogenous TLR. Coud you please tell us your suggestion?
Functional activity of this cell line requires co-transfection of MD-2 and CD14. With regards to readout assays, we have evaluated both the NBP2-26268 (TLR4) and NBP2-26266 (TLR2) with the SEAP reporter assays as shown on the respective data sheets. We don't perform semi-quantitative PCR and I am sorry we don't have any information regarding the ligand the customer used nor a Defencin PCR quantitative PCR readout assay. Hence, it would be difficult to comment further on the customer's results. We would suggest that the customer use the techniques and assays shown on the data sheet as positive controls for cell line function. This includes the SEAP reporter assays which require co-transfection of the NF-kB SEAP reporter, and for the TLR4 cell it also requires co-transfection of MD2 and CD14. This also includes using established TLR4 (LPS) and TLR2 (PAM3CSK4). Once the researcher establishes the positive control system, they can move to testing their ligand with the SEAP readout assay and then to their Defencin readout assay. However, it must be recognized that the suitability of the cell lines for a researcher's model system needs to be empirically determined and optimized by the researcher. Both the NBP2-26266 and NBP2-26268 cell lines are selected for function as assayed by a SEAP reporter assay (see data on data sheets). Overexpression results are shown by flow cytometry using the appropriate TLR antibodies with respect to the Vector control cell line. Please refer to the data sheets for these results. For WB, we use an HA Tag antibody. The TLRs were not recognized by WB using the TLR antibodies we evaluated. We would suggest using a Tag antibody if the researcher wishes to detect the TLR constructs expressed by the cell lines. We are not studying endogenous TLRs in the cell lines, the focus is on the functional aspects of the TLR genes stably transfected into the cell lines. If the customer wishes to use TLR antibodies to assess the TLRs, then flow cytometry assays should be performed as shown on the data sheets. On another note, HEK293 do not express functional TLR2 or TLR4. I am not sure what ligand the researcher is using but activation of normal HEK293 cells might be occurring through other pathways.
Does the TLR4 cell line express the related factors (MD2, CD14 and LBP). Has expression of each gene been confirmed?
We have not evaluated the gene expression of MD2, CD14 or LBP in this cell line. The parent cell line is HEK293 as denoted in the data sheet. One would expect that the gene expression profile would be similar to the HEK293 parent cell line. There is considerable information in the scientific literature about HEK293. We have not evaluated the gene expression profile of the parent HEK293 cell line in our hands.
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