TLR12 Stable Cell Line

Description（描述）

The TLR12 stable cell line is a stably transfected cell line which expresses full-length mouse Toll-like receptor 12 (TLR12) with an N-terminal HA tag. TLR12 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 TLR12 stable line is blasticidin.

Application（应用）

The TLR12 stable cell line can be used for TLR12 flow cytometric calibration, detection control and TLR12-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 TLR12 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 TLR12 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 TLR12 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 TLR12 expression in the TLR12 stable cell line using an HA antibody (20 ug total protein/lane). Legend. Vect: Vector control stable cell line (IML-200); TLR12: TLR12 stable cell line (IML-212).

Figure 2. Expression of TLR12 in the TLR12 stable cell line was analyzed by flow cytometry using a FITC-conjugated TLR12 antibody (IMG-5034C) 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. Victoria J Philbin, Muhammad Iqbal, Yvonne Boyd, Marianne J Goodchild, Richard K Beal, Nat Bumstead, John Young, Adrian L Smith. Identification and characterization of a functional, alternatively spliced Toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens. Immunology. 2005 April; 114(4): 507–521.
2. Wendy W C van Maren, Joannes F M Jacobs, I Jolanda M de Vries, Stefan Nierkens, Gosse J Adema. Toll-like receptor signalling on Tregs: to suppress or not to suppress? Immunology. 2008 August; 124(4): 445–452.
3. Vida A. Dennis, Saurabh Dixit, Shannon M. OBrien, Xavier Alvarez, Bapi Pahar, Mario T. Philipp. Live Borrelia burgdorferi Spirochetes Elicit Inflammatory Mediators from Human Monocytes via the Toll-Like Receptor Signaling Pathway. Infect Immun. 2009 March; 77(3): 1238–1245.
4. Crystal Morales, Shuang Wu, Yi Yang, Bing Hao, Zihai Li. Drosophila Glycoprotein 93 Is an Ortholog of Mammalian Heat Shock Protein gp96 (grp94, HSP90b1, HSPC4) and Retains Disulfide Bond-Independent Chaperone Function for TLRs and Integrins. J Immunol. 2009 October 15; 183(8): 5121–5128.
5. Erin L. Lousberg, Kerrilyn R. Diener, Cara K. Fraser, Simon Phipps, Paul S. Foster, Weisan Chen, Satoshi Uematsu, Shizuo Akira, Sarah A. Robertson, Michael P. Brown, John D. Hayball. Antigen-Specific T-Cell Responses to a Recombinant Fowlpox Virus Are Dependent on MyD88 and Interleukin-18 and Independent of Toll-Like Receptor 7 (TLR7)- and TLR9-Mediated Innate Immune Recognition. J Virol. 2011 April; 85(7): 3385–3396.
6. Nina Gratz, Harald Hartweger, Ulrich Matt, Franz Kratochvill, Marton Janos, Stefanie Sigel, Barbara Drobits, Xiao-Dong Li, Sylvia Knapp, Pavel Kovarik. Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection. PLoS Pathog. 2011 May; 7(5): e1001345.
7. Dong-Mi Shin, Chang-Hoon Lee, Herbert C. Morse, III. IRF8 Governs Expression of Genes Involved in Innate and Adaptive Immunity in Human and Mouse Germinal Center B Cells. PLoS One. 2011; 6(11): e27384.
8. Norio Matsushima, Takanori Tanaka, Purevjav Enkhbayar, Tomoko Mikami, Masae Taga, Keiko Yamada, Yoshio Kuroki. Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genomics. 2007; 8: 124.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-212	TLR12 Stably Transfected HEK 293 Cells	imgenex	1Vial	13328	2-3周