TLR9 Stable Cell Line

Description（描述）

The TLR9 stable cell line is a stably transfected HEK 293 line which expresses full-length human Toll-like receptor 9 (TLR9) with no epitope tag. TLR9 expression in this cell line has been validated by flow cytometry (Fig. 1). Functional activity of this cell line has been validated using the NF-kB/SEAPorter™ Assay Kit (IMK-515) (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 TLR9 stable cell line is blasticidin.

Application（应用）

The TLR9 stable cell line can be used for TLR9 flow cytometric calibration and detection control as well as TLR9-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 TLR9 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 TLR9 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 TLR9 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. Flow analysis of TLR9 expression in the TLR9 stable cell line. Intracellular expression of TLR9 in the TLR9 stable cell line was analyzed by flow cytometry using a PE-conjugated TLR9 antibody (IMG-305D) and compared with the Vector control stable cell line (IML-200). IMGENEX’s Intracellular TLR Staining Flow Kit (10098K) was used for this test.

Figure 2. Functional analysis of the TLR9 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 TLR9 stable cell line (IML-209) were transfected with NF-kB/SEAP reporter plasmid for 16 h. Cells were stimulated with various amounts of CpG (IMG-2209Hpt) for 24 h followed by SEAP assay.

Reference（参考文献）
1. Stanimir Ivanov, Ana-Maria Dragoi, Xin Wang, Corrado Dallacosta, Jennifer Louten, Giovanna Musco, Giovanni Sitia, George S. Yap, Yinsheng Wan, Christine A. Biron, Marco E. Bianchi, Haichao Wang, Wen-Ming Chu. A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA. Blood. 2007 September 15; 110(6): 1970–1981.
2. Liqin Zheng, Nicole Asprodites, Angela H. Keene, Paulo Rodriguez, Kevin D. Brown, Eduardo Davila. TLR9 engagement on CD4 T lymphocytes represses γ-radiation–induced apoptosis through activation of checkpoint kinase response elements. Blood. 2008 March 1; 111(5): 2704–2713.
3. Annapoorani Chockalingam, James C. Brooks, Jody L. Cameron, Lisa K. Blum, Cynthia A. Leifer. TLR9 traffics through the Golgi complex to localize to endolysosomes and respond to CpG DNA. Immunol Cell Biol. 2009 Mar–Apr; 87(3): 209–217.
4. Toshihiro Ito, Matthew Schaller, Tracy Raymond, Amrita D. Joshi, Ana L. Coelho, Fabiani G. Frantz, William F. Carson, IV, Cory M. Hogaboam, Nicholas W. Lukacs, Theodore J. Standiford, Sem H. Phan, Stephen W. Chensue, Steven L. Kunkel. Toll-like Receptor 9 Activation Is a Key Mechanism for the Maintenance of Chronic Lung Inflammation. Am J Respir Crit Care Med. 2009 December 15; 180(12): 1227–1238.
5. Tobias Haas, Frank Schmitz, Antje Heit, Hermann Wagner. Sequence independent interferon-α induction by multimerized phosphodiester DNA depends on spatial regulation of Toll-like receptor-9 activation in plasmacytoid dendritic cells. Immunology. 2009 February; 126(2): 290–298.
6. Natália B. Carvalho, Fernanda S. Oliveira, Fernanda V. Durães, Leonardo A. de Almeida, Manuela Flórido, Luana O. Prata, Marcelo V. Caliari, Rui Appelberg, Sérgio C. Oliveira. Toll-Like Receptor 9 Is Required for Full Host Resistance to Mycobacteriumavium Infection but Plays No Role in Induction of Th1 Responses. Infect Immun. 2011 April; 79(4): 1638–1646.

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