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TLR4/MD-2/CD14/NF-kB/SEAP穩(wěn)轉(zhuǎn)報(bào)告細(xì)胞株
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TLR4/MD-2/CD14/NF-kB/SEAP Stable Reporter Cell Line
Description（描述）
The TLR4 / MD-2 / CD14 reporter cell line is a stably co-transfected cell line which expresses full-length human Toll-like receptor 4 （TLR4）, human MD-2, human CD14 and secreted alkaline phosphatase （SEAP） reporter genes under the transcriptional control of an NF-kB response element. The TLR4 / MD-2 / CD14 reporter cell line has been validated by a ligand dose response assay （Figure 1）.
IMGENEX offers the TLR4/MD-2/CD14/NF-kB Reporter Assay as a service.
Complete Growth Medium（*培養(yǎng)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 + 2 ug/ml puromycin + 200 ug/ml zeocin + 500 ug/ml G418 （Geneticin）.
Note: The selection agents for this cell line are blasticidin, puromycin, zeocin and G418.
Application（應(yīng)用）
The TLR4 / MD-2 / CD14 reporter line can be used for TLR4-dependent functional assays as well as screening of TLR4 agonists or antagonists.
Product Handling Protocol（產(chǎn)品處理協(xié)議）
Note: Please read the entire data sheet before thawing. It is recommended that users follow good tissue culture practice. The TLR4 / MD-2 cell line is 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 TLR4 reporter 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 reporter cells at this stage without any selection agents.
7. Transfer the TLR4 / MD-2 cell 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 TLR4 / MD-2 cell 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.
1. Prepare a sterile 15-ml tube with 9 ml fresh medium without selection agents pre-warmed at 37oC.
2. Thaw the TLR4 reporter 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 reporter cells at this stage without any selection agents.
7. Transfer the TLR4 / MD-2 cell 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 TLR4 / MD-2 cell 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（安全注意事項(xiàng)）
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 TLR4 / MD-2 /CD14 line.
- Wash hands after handling the TLR4 / MD-2 /CD14 line and before leaving the lab.
- Decontaminate work surface with disinfectant or 70% ethanol before and after working with the TLR4 / MD-2 /CD14 reporter line.
- All waste should be considered hazardous.
- Dispose of all liquid waste after each experiment and treat with bleach.
Figure 1. Evaluation of the functional activity of the TLR4 / MD-2 / CD14 cell line by ligand dose response assay. TLR4 / MD-2 / CD14 reporter cell line （IML-104MD2CD14） was plated in 96-well plates at 1 x 10^5 cells/well. After 16 h, cells were stimulated with various amount of LPS （IMG-2204） for 24 h. SEAP was analyzed using the SEAPorter Assay Kit （10055K）. A. LPS-mediated TLR4 activation, as measured by SEAP activity, was increased in a dose dependent manner. B. The values from （A） were used to determine the EC50 of LPS. The EC50, or the half maximal activity concentration, represents the concentration of LPS that was required for 50% activation of TLR4 as measured by SEAP activity.
Data Summary: The IML-104MD2CD14 cell line was activated by LPS in a dose-response manner, of which EC50 was measured as 8.98 ng/ml.
Reference（參考文獻(xiàn)）
1. Kou-Gi Shyu, Bao-Wei Wang, Chiu-Mei Lin, Hang Chang. Cyclic stretch enhances the expression of Toll-like Receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-κB pathway.J Biomed Sci. 2010; 17（1）: 15.
2. Huan Yang, Huiqin Zhou, Ping Feng, Xiaoni Zhou, Huiyan Wen, Xiaofang Xie, Haiying Shen, Xueming Zhu. Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion. J Exp Clin Cancer Res. 2010; 29（1）: 92.
3. Iain Martin Sheldon, Mark H. Roberts. Toll-Like Receptor 4 Mediates the Response of Epithelial and Stromal Cells to Lipopolysaccharide in the Endometrium. PLoS One. 2010; 5（9）: e12906.
4. Andreas Schaeffler, Philipp Gross, Roland Buettner, Cornelius Bollheimer, Christa Buechler, Markus Neumeier, Andrea Kopp, Juergen Schoelmerich, Werner Falk. Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-κB pathway in adipocytes links nutritional signalling with innate immunity. Immunology. 2009 February; 126（2）: 233–245.
5. J. Jason Hoth, Jonathan D. Wells, Noel A. Brownlee, Elizabeth M. Hiltbold, J. Wayne Meredith, Charles E. McCall, Barbara K. Yoza. Toll-like receptor 4 dependent responses to lung injury in a murine model of pulmonary contusion. Shock. 2009 April; 31（4）: 376–381.
6. Qing-Wu Yang, Feng-Lin Lu, Yu Zhou, Lin Wang, Qi Zhong, Sen Lin, Jing Xiang, Jing-Cheng Li, Chuan-Qing Fang, Jing-Zhou Wang. HMBG1 mediates ischemia–reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. J Cereb Blood Flow Metab. 2011 February; 31（2）: 593–605.
2. Huan Yang, Huiqin Zhou, Ping Feng, Xiaoni Zhou, Huiyan Wen, Xiaofang Xie, Haiying Shen, Xueming Zhu. Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion. J Exp Clin Cancer Res. 2010; 29（1）: 92.
3. Iain Martin Sheldon, Mark H. Roberts. Toll-Like Receptor 4 Mediates the Response of Epithelial and Stromal Cells to Lipopolysaccharide in the Endometrium. PLoS One. 2010; 5（9）: e12906.
4. Andreas Schaeffler, Philipp Gross, Roland Buettner, Cornelius Bollheimer, Christa Buechler, Markus Neumeier, Andrea Kopp, Juergen Schoelmerich, Werner Falk. Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-κB pathway in adipocytes links nutritional signalling with innate immunity. Immunology. 2009 February; 126（2）: 233–245.
5. J. Jason Hoth, Jonathan D. Wells, Noel A. Brownlee, Elizabeth M. Hiltbold, J. Wayne Meredith, Charles E. McCall, Barbara K. Yoza. Toll-like receptor 4 dependent responses to lung injury in a murine model of pulmonary contusion. Shock. 2009 April; 31（4）: 376–381.
6. Qing-Wu Yang, Feng-Lin Lu, Yu Zhou, Lin Wang, Qi Zhong, Sen Lin, Jing Xiang, Jing-Cheng Li, Chuan-Qing Fang, Jing-Zhou Wang. HMBG1 mediates ischemia–reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. J Cereb Blood Flow Metab. 2011 February; 31（2）: 593–605.
產(chǎn)品報(bào)價(jià)：
| 貨號(hào) | 名稱 | 產(chǎn)地 | 規(guī)格 | 報(bào)價(jià)/元 | 貨期 |
| IML-104/MD2CD14 | TLR4/MD-2/CD14/NF-kB/SEAP Stable Reporter Cell Line | imgenex | 1Vial | 19111 | 2-3周 |
