TLR10 Stable Cell Line

Description（描述）

The TLR10 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 10 (TLR10) with an N-terminal HA tag. TLR10 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 TLR10 stable line is blasticidin.

Application（应用）

The TLR10 stable cell line can be used for TLR10 flow cytometric calibration and detection control.

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 TLR10 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 TLR10 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 TLR10 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 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 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 TLR10 expression in the TLR10 stable cell line using an HA antibody (20 ug total protein/lane). Legend. Vect: Vector control stable cell line (IML-200); TLR10: TLR10 stable cell line (IML-210).

Figure 2. Flow analysis of TLR10 expression in the TLR10 stable cell line. Cell surface expression of TLR10 in the TLR10 stable cell line was analyzed by flow cytometry using a FITC-conjugated TLR10 antibody and compared with the Vector control stable cell line (IML-200). IMGENEX’s mouse IgG1 isotype control (20105) and Cell Surface TLR Staining Flow Kit (10099K) were used for this test. *Note: An endogenous basal level of TLR10 is present in HEK 293 cells.

Reference（参考文献）
1. Martijn D. de Kruif, Tatty E. Setiati, Albertus T. A. Mairuhu, Penelopie Koraka, Hella A. Aberson, C. Arnold Spek, Albert D. M. E. Osterhaus, Pieter H. Reitsma, Dees P. M. Brandjes, Augustinus Soemantri, Eric C. M. van Gorp. Differential Gene Expression Changes in Children with Severe Dengue Virus Infections. PLoS Negl Trop Dis. 2008 April; 2(4): e215.
2. Cheng Luo, Xiangjun Yang, Angela D. Kain, David W. Powell, Markus H. Kuehn, Gülgün Tezel. Glaucomatous Tissue Stress and the Regulation of Immune Response through Glial Toll-like Receptor Signaling. Invest Ophthalmol Vis Sci. 2010 November; 51(11): 5697–5707.
3. Gabriele Köllisch, Behnam Naderi Kalali, Verena Voelcker, Reinhard Wallich, Heidrun Behrendt, Johannes Ring, Stefan Bauer, Thilo Jakob, Martin Mempel, Markus Ollert. Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes. Immunology. 2005 April; 114(4): 531–541.
4. Anne Månsson, Mikael Adner, Ulf Höckerfelt, Lars-Olaf Cardell. A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by Pam3CSK4, R-837 and CpG-2006 stimulation. Immunology. 2006 August; 118(4): 539–548.
5. Helen KW Law, Chung Yan Cheung, Sin Fun Sia, Yuk On Chan, JS Malik Peiris, Yu Lung Lau. Toll-like receptors, chemokine receptors and death receptor ligands responses in SARS coronavirus infected human monocyte derived dendritic cells. BMC Immunol. 2009; 10: 35.
6. Rajiv Gandhi Govindaraj, Balachandran Manavalan, Gwang Lee, Sangdun Choi. Molecular Modeling-Based Evaluation of hTLR10 and Identification of Potential Ligands in Toll-Like Receptor Signaling. PLoS One. 2010; 5(9): e12713.
7. Luis B. Barreiro, Meriem Ben-Ali, Hélène Quach, Guillaume Laval, Etienne Patin, Joseph K. Pickrell, Christiane Bouchier, Magali Tichit, Olivier Neyrolles, Brigitte Gicquel, Judith R. Kidd, Kenneth K. Kidd, Alexandre Alcaïs, Josiane Ragimbeau, Sandra Pellegrini, Laurent Abel, Jean-Laurent Casanova, Lluís Quintana-Murci. Evolutionary Dynamics of Human Toll-Like Receptors and Their Different Contributions to Host Defense. PLoS Genet. 2009 July; 5(7): e1000562.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-210	TLR10 Stably Transfected HEK 293 Cells	imgenex	1Vial	13328	2-3周