TLR5 Stable Cell Line

Description（描述）

TLR5 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 5 (TLR5) with an N-terminal HA tag. TLR5 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/SEAPorter™ Assay Kit (IMK-515, Fig. 3).

Complete Growth Medium（完全培养基）

DMEM with 4.5 g/L glucose + 10% FBS + 4 mM L-glutamine + 1 mM sodium pyruvate + 100 units/ml penicillin + 100 ug/ml streptomycin + 10 ug/ml blasticidin.
Note: The selection agent for the TLR5 stable line is blasticidin.

Application（应用）

The TLR5 stable cell line can be used for TLR5 flow cytometric calibration and detection control as well as TLR5-dependent functional assays.

Product Handling Protocol（产品处理协议）

Note: Please read the entire data sheet before thawing. It is recommended that users follow good tissue culture practice. The stable cells are sterile and all work should be performed under sterile conditions.
1. Prepare a sterile 15-ml tube with 9 ml fresh medium without selection agents pre-warmed at 37oC.
2. Thaw the TLR5 stable cell line vial quickly in a 37oC water bath, keeping the cap portion out of the water to avoid any possible contamination.
3. Upon thawing, take the vial out of the water and clean it with 70% ethanol to decontaminate.
4. Transfer contents to the 15-ml tube (Step 1) and mix with medium by gentle inversion of tube.
5. Centrifuge at 1,000 RPM for 5 minutes.
6. Remove supernatant and resuspend pellet in 10 ml of fresh medium without selection agents.
Note: It is important to grow the stable cells at this stage without any selection agents.
7. Transfer the TLR5 stable line into a 25-cm2 tissue culture flask and incubate at 37oC in a 95% air-5% CO2 mixture.
8. After cells settle down (in 1-3 days), remove the medium and replace with fresh complete growth medium containing selection agents.
9. At 70-80% confluency, detach the cells by trypsinization and split into new flasks with fresh complete growth medium.
10. Freeze the TLR5 stable line at 3~4 x 10^6 cells/ml per cryogenic vial. For optimal viability after freezing, freeze cells when they have reached log phase growth (95-98% confluency). Detach by trypsinization at 37oC for 5 min, and harvest by mixing with 3 volumes of fresh medium followed by centrifugation (Step 5). Resuspend the pellet in freeze media (FBS with 10% DMSO). Add suspension to cryogenic vials in 1 ml aliquots. Place cryogenic vials, in a tissue culture approved cryogenic vial container, in -80oC freezer for 24-48 hours. After 24-48 hours, move the vials into liquid nitrogen storage.

Safety Considerations

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.

Figure 1. Western blot analysis of TLR5 expression in the TLR5 stable cell line using an HA antibody (20 ug total protein/lane). Legend. Vect: Vector control stable cell line (IML-200); TLR5: TLR5 stable cell line.
Figure 2. Flow analysis of TLR5 expression in the TLR5 stable cell line. Cell surface expression of TLR5 in the TLR5 stable cell line was analyzed by flow cytometry using an Alexa Fluor® 647-conjugated TLR5 antibody (IMG-664AF647) and compared with the Vector control stable cell line (IML-200). A mouse IgG2a isotype control and IMGENEX’s Cell Surface TLR Staining Flow Kit (10099K) were used for this test. *Note: An endogenous level of TLR5 is present in HEK 293 cells.

Figure 3. Functional analysis of the TLR5 stable cell line. The assay was performed using the NF-kB SEAPorter™ Assay Kit (IMK-515). The Vector control stable cell line (IML-200) and TLR5 stable cell line (IML-205) were transfected with NF-kB/SEAP reporter plasmid for 16 h. Cells were stimulated with Flagellin (IMG-2205) for 24 h followed by SEAP assay.

Reference（参考文献）
1. Zhe Zhang, William Reenstra, Daniel J. Weiner, Jean-Pierre Louboutin, James M. Wilson. The p38 Mitogen-Activated Protein Kinase Signaling Pathway Is Coupled to Toll-Like Receptor 5 To Mediate Gene Regulation in Response to Pseudomonas aeruginosa Infection in Human Airway Epithelial Cells. Infect Immun. 2007 December; 75(12): 5985–5992.
2. N Oshima, S Ishihara, M A K Rumi, M M Aziz, Y Mishima, C Kadota, I Moriyama, N Ishimura, Y Amano, Y Kinoshita. A20 is an early responding negative regulator of Toll-like receptor 5 signalling in intestinal epithelial cells during inflammation. Clin Exp Immunol. 2010 February; 159(2): 185–198.
3. Matam Vijay-Kumar, Jesse D. Aitken, Amrita Kumar, Andrew S. Neish, Satoshi Uematsu, Shizuo Akira, Andrew T. Gewirtz. Toll-Like Receptor 5-Deficient Mice Have Dysregulated Intestinal Gene Expression and Nonspecific Resistance to Salmonella-Induced Typhoid-Like Disease. Infect Immun. 2008 March; 76(3): 1276–1281.
4. Sang Hoon Rhee, Eunok Im, Charalabos Pothoulakis. Toll-like receptor 5 engagement modulates tumor development and growth in a mouse xenograft model of human colon cancer. Gastroenterology. 2008 August; 135(2): 518–528.
5. Amy E. Morris, H. Denny Liggitt, Thomas R. Hawn, Shawn J. Skerrett. Role of Toll-like receptor 5 in the innate immune response to acute P. aeruginosa pneumonia. Am J Physiol Lung Cell Mol Physiol. 2009 December; 297(6): L1112–L1119.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-205	TLR5 Stably Transfected HEK 293 Cells	imgenex	1Vial	13328	2-3周