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產品簡介
產品介紹
TLR4 Stable Cell Line
Description（描述）
The TLR4 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 4 （TLR4） with an N-terminal HA tag. TLR4 expression in this stable cell line has been validated by Western blotting （Fig. 1） and flow cytometry （Fig. 2）. Functional activity of this stable cell line has been validated by the NF-kB/SEAPorter™ Assay Kit （IMK-515, Fig. 3）.
Complete Growth Medium（*培養基）
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 TLR4 stable line is blasticidin.
Note: The selection agent for the TLR4 stable line is blasticidin.
Application（應用）
The TLR4 stable cell line can be used for TLR4 flow cytometric calibration and detection control as well as TLR4-dependent functional assays.
Product Handling Protocol（產品處理協議）
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 TLR4 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 TLR4 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 TLR4 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.
1. Prepare a sterile 15-ml tube with 9 ml fresh medium without selection agents pre-warmed at 37oC.
2. Thaw the TLR4 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 TLR4 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 TLR4 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（安全注意事項）
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 cells.
• All waste should be considered hazardous.
• Dispose of all liquid waste after each experiment and treat with bleach.
• 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 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 TLR4 expression in the TLR4 stable cell line using an HA antibody （20 ug total protein/lane）. Legend. Vect: Vector control stable cell line （IML-200）; TLR4: TLR4 stable cell line （IML-204）.
Figure 2. Flow analysis of TLR4 expression in the TLR4 stable cell line. Cell surface expression of TLR4 in the TLR4 stable cell line （IML-204） was analyzed by flow cytometry using a FITC-conjugated TLR4 antibody （IMG-417C） and compared with the Vector control stable cell line （IML-200）. IMGENEX’s Cell Surface TLR Staining Flow Kit （10099K） was used for this test.
Figure 3. Functional analysis of the TLR4 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 TLR4 stable cell line （IML-204） were co-transfected with NF-kB/SEAP reporter plasmid and MD-2/CD14 expression plasmid for 16 h. Cells were stimulated with LPS （IMG-2204） for 24 h followed by SEAP assay.
Reference（參考文獻）
1. Matam Vijay-Kumar, Jesse D Aitken, Frederic A Carvalho, Thomas R Ziegler, Andrew T Gewirtz, Vijay Ganji. Loss of function mutation in toll-like receptor-4 does not offer protection against obesity and insulin resistance induced by a diet high in trans fat in mice. J Inflamm （Lond） 2011; 8: 2.
2. Humberto M Garay-Malpartida, Roberta F Mourão, Marluce Mantovani, Icaro A Santos, Mari C Sogayar, Anna C Goldberg. Toll-like receptor 4 （TLR4） expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis. BMC Immunol. 2011; 12: 18.
3. C. Dirk Keene, Rubens Chang, Christina Stephen, Mary Nivison, Samuel E. Nutt, Amy Look, Richard M. Breyer, Phillip J. Horner, Robert Hevner, Thomas J. Montine. Protection of Hippocampal Neurogenesis from Toll-Like Receptor 4-Dependent Innate Immune Activation by Ablation of Prostaglandin E2 Receptor Subtype EP1 or EP2. Am J Pathol. 2009 June; 174（6）: 2300–2309.
4. Giovanna Zanoni, Riccardo Navone, Claudio Lunardi, Giuseppe Tridente, Caterina Bason, Simona Sivori, Ruggero Beri, Marzia Dolcino, Enrico Valletta, Roberto Corrocher, Antonio Puccetti. In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes. PLoS Med. 2006 September; 3（9）: e358.
5. Elizabeth M. Lawlor, Magali M. Moretto, Imtiaz A. Khan. Optimal CD8 T-Cell Response against Encephalitozoon cuniculi Is Mediated by Toll-Like Receptor 4 Upregulation by Dendritic Cells. Infect Immun. 2010 July; 78（7）: 3097–3102.
1. Matam Vijay-Kumar, Jesse D Aitken, Frederic A Carvalho, Thomas R Ziegler, Andrew T Gewirtz, Vijay Ganji. Loss of function mutation in toll-like receptor-4 does not offer protection against obesity and insulin resistance induced by a diet high in trans fat in mice. J Inflamm （Lond） 2011; 8: 2.
2. Humberto M Garay-Malpartida, Roberta F Mourão, Marluce Mantovani, Icaro A Santos, Mari C Sogayar, Anna C Goldberg. Toll-like receptor 4 （TLR4） expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis. BMC Immunol. 2011; 12: 18.
3. C. Dirk Keene, Rubens Chang, Christina Stephen, Mary Nivison, Samuel E. Nutt, Amy Look, Richard M. Breyer, Phillip J. Horner, Robert Hevner, Thomas J. Montine. Protection of Hippocampal Neurogenesis from Toll-Like Receptor 4-Dependent Innate Immune Activation by Ablation of Prostaglandin E2 Receptor Subtype EP1 or EP2. Am J Pathol. 2009 June; 174（6）: 2300–2309.
4. Giovanna Zanoni, Riccardo Navone, Claudio Lunardi, Giuseppe Tridente, Caterina Bason, Simona Sivori, Ruggero Beri, Marzia Dolcino, Enrico Valletta, Roberto Corrocher, Antonio Puccetti. In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes. PLoS Med. 2006 September; 3（9）: e358.
5. Elizabeth M. Lawlor, Magali M. Moretto, Imtiaz A. Khan. Optimal CD8 T-Cell Response against Encephalitozoon cuniculi Is Mediated by Toll-Like Receptor 4 Upregulation by Dendritic Cells. Infect Immun. 2010 July; 78（7）: 3097–3102.
訂購信息：
| 貨號 | 名稱 | 產地 | 規格 | 報價/元 | 貨期 |
| IML-204 | TLR4 Stably Transfected HEK 293 Cells | imgenex | 1Vial | 13328 | 2-3周 |
