TLR11 Stable Cell Line

Description（描述）

The TLR11 stable cell line is a stably transfected cell line which expresses full-length mouse Toll-like receptor 11 (TLR11) with an N-terminal HA tag. TLR11 expression in this cell line has been validated by Western blotting (Fig. 1) and flow cytometry (Fig. 2).

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 TLR11 stable line is blasticidin.

Application（应用）

The TLR11 stable cell line can be used for TLR11 flow cytometric calibration, detection control and TLR11-based functional studies.

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 TLR11 stable 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 TLR11 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 TLR11 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（安全注意事项）

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 cell line.
• Wash hands after handling the stable cell line and before leaving the lab.
• Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable 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 TLR11 expression in the TLR11 stable cell line using an HA antibody (20 ug total protein/lane). Legend. Vect: Vector control stable cell line (IML-200); TLR11: TLR11 stable cell line (IML-211).

Figure 2.  Expression of TLR11 in the TLR11 stable cell line was analyzed by flow cytometry using a FITC-conjugated TLR11 antibody (IMG-5104C) and compared with the Vector control stable cell line (IML-200). Flow samples were prepared using IMGENEX’s Intracellular TLR Staining Flow Kit (10098K).

Reference（参考文献）
1. Sergey S. Seregin, Yasser A. Aldhamen, Daniel M. Appledorn, Charles F. Aylsworth, Sarah Godbehere, Chyong-Jy Joyce Liu, Dionisia Quiroga, Andrea Amalfitano. TRIF Is a Critical Negative Regulator of TLR Agonist Mediated Activation of Dendritic Cells In Vivo. PLoS One. 2011; 6(7): e22064.
2. Myriam Baratin, Sophie Roetynck, Catherine Lépolard, Christine Falk, Serge Sawadogo, Satoshi Uematsu, Shizuo Akira, Bernhard Ryffel, Jean-Gérard Tiraby, Lena Alexopoulou, Carsten J. Kirschning, Jürg Gysin, Eric Vivier, Sophie Ugolini. Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum. Proc Natl Acad Sci U S A. 2005 October 11; 102(41): 14747–14752.
3. Kaury Kucera, A. Alicia Koblansky, Lauren P. Saunders, Kendra B. Frederick, Enrique M. De La Cruz, Sankar Ghosh, Yorgo Modis. Structure-Based Analysis of Toxoplasma gondii Profilin: A Parasite-Specific Motif is Required for Recognition by Toll-like Receptor 11. J Mol Biol. 2010 November 5; 403(4): 616–629.
4. Bibhuti B Mishra, Uma Mahesh Gundra, Judy M Teale. Expression and distribution of Toll-like receptors 11–13 in the brain during murine neurocysticercosis. J Neuroinflammation. 2008; 5: 53.
5. Reed Pifer, Alicia Benson, Carolyn R. Sturge, Felix Yarovinsky. UNC93B1 Is Essential for TLR11 Activation and IL-12-dependent Host Resistance to Toxoplasma gondii. J Biol Chem. 2011 February 4; 286(5): 3307–3314.
6. Zhenyu Cai, Zhongcheng Shi, Amir Sanchez, Tingting Zhang, Mingyao Liu, Jianghua Yang, Fen Wang, Dekai Zhang. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells. J Biol Chem. 2009 November 27; 284(48): 33088–33096.
7. Woraporn Sukhumavasi, Charlotte E. Egan, Amy L. Warren, Gregory A. Taylor, Barbara A. Fox, David J. Bzik, Eric Y. Denkers. Toll-Like Receptor Adaptor MyD88 is Essential for Pathogen Control During Oral Toxoplasma gondii Infection but not Adaptive Immunity Induced by a Vaccine Strain of the Parasite. J Immunol. 2008 September 1; 181(5): 3464–3473.
8. Zhongcheng Shi, Zhenyu Cai, Amir Sanchez, Tingting Zhang, Shu Wen, Jun Wang, Jianhua Yang, Songbin Fu, Dekai Zhang. A Novel Toll-like Receptor That Recognizes Vesicular Stomatitis Virus. J Biol Chem. 2011 February 11; 286(6): 4517–4524.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-211	TLR11 Stably Transfected HEK 293 Cells	imgenex	1Vial	13328	2-3周