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產品簡介
產品介紹
TLR2 Stable Cell Line
Description（描述）
The TLR2 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 2 （TLR2） with an N-terminal HA tag. TLR2 expression in these cells has been validated by Western blotting （Fig. 1） and flow cytometry （Fig. 2）. Functional activity of this cell line has been validated using the NF-kB/SEAPorter™ Assay Kit （IMGENEX, 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 TLR2 stable line is blasticidin.
Note: The selection agent for the TLR2 stable line is blasticidin.
Application（應用）
The TLR2 stable cell line can be used for TLR2 flow cytometric calibration and detection control as well as TLR2-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 TLR2 stable cells 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 TLR2 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 TLR2 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 TLR2 stable 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 TLR2 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 TLR2 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 TLR2 stable 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）
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 TLR2 stable line.
• Wash hands after handling the TLR2 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 TLR2 stable line.
• Wash hands after handling the TLR2 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 TLR2 expression in the TLR2 stable cell line using an HA antibody （20 ug total protein/lane）. Legend. Vect: Vector/HEK 293 （IMGENEX, IML-200）; TLR2: TLR2 stable cell line （IML-202）.
Figure 2. Surface expression of TLR2 in the TLR2 stable cell line was analyzed by flow cytometry using a PE-conjugated TLR2 antibody （IMG-416D） and compared with the Vector/HEK 293 control （IML-200）. IMGENEX’s Surface TLR Staining Flow Kit （10099K） was used for this test.
Reference（參考文獻）
1. Mohsin Md. Syed, Nirmal K. Phulwani, Tammy Kielian. Tumor necrosis factor-alpha （TNF-α） regulates Toll-like receptor 2 （TLR2） expression in microglia. J Neurochem. 2007 November; 103（4）: 1461–1471.
2. Allison L. van Lint, Matthew R. Murawski, Rory E. Goodbody, Martina Severa, Katherine A. Fitzgerald, Robert W. Finberg, David M. Knipe, Evelyn A. Kurt-Jones. Herpes Simplex Virus Immediate-Early ICP0 Protein Inhibits Toll-Like Receptor 2-Dependent Inflammatory Responses and NF-κB Signaling. J Virol. 2010 October; 84（20）: 10802–10811.
3. Louise Hjerrild Zeuthen, Lisbeth Nielsen Fink, Hanne Frøkiær. Toll-like receptor 2 and nucleotide-binding oligomerization domain-2 play divergent roles in the recognition of gut-derived lactobacilli and bifidobacteria in dendritic cells. Immunology. 2008 August; 124（4）: 489–502.
4. Jinkeng Asong, Margreet A. Wolfert, Kaustabh K. Maiti, Douglas Miller, Geert-Jan Boons. Binding and Cellular Activation Studies Reveal That Toll-like Receptor 2 Can Differentially Recognize Peptidoglycan from Gram-positive and Gram-negative Bacteria. J Biol Chem. 2009 March 27; 284（13）: 8643–8653.
5. Jian-Wen Dong, Jesus G. Vallejo, Huei-Ping Tzeng, James A Thomas, Douglas L. Mann. Innate immunity mediates myocardial preconditioning through Toll-like receptor 2 and TIRAP-dependent signaling pathways. Am J Physiol Heart Circ Physiol. 2010 March; 298（3）: H1079–H1087.
6. Alexandre Jacques, Christian Bleau, Claire Turbide, Nicole Beauchemin, Lucie Lamontagne. Macrophage interleukin-6 and tumour necrosis factor-α are induced by coronavirus fixation to Toll-like receptor 2/heparan sulphate receptors but not carcinoembryonic cell adhesion antigen 1a. Immunology. 2009 September; 128（1pt2）: e181–e192.
1. Mohsin Md. Syed, Nirmal K. Phulwani, Tammy Kielian. Tumor necrosis factor-alpha （TNF-α） regulates Toll-like receptor 2 （TLR2） expression in microglia. J Neurochem. 2007 November; 103（4）: 1461–1471.
2. Allison L. van Lint, Matthew R. Murawski, Rory E. Goodbody, Martina Severa, Katherine A. Fitzgerald, Robert W. Finberg, David M. Knipe, Evelyn A. Kurt-Jones. Herpes Simplex Virus Immediate-Early ICP0 Protein Inhibits Toll-Like Receptor 2-Dependent Inflammatory Responses and NF-κB Signaling. J Virol. 2010 October; 84（20）: 10802–10811.
3. Louise Hjerrild Zeuthen, Lisbeth Nielsen Fink, Hanne Frøkiær. Toll-like receptor 2 and nucleotide-binding oligomerization domain-2 play divergent roles in the recognition of gut-derived lactobacilli and bifidobacteria in dendritic cells. Immunology. 2008 August; 124（4）: 489–502.
4. Jinkeng Asong, Margreet A. Wolfert, Kaustabh K. Maiti, Douglas Miller, Geert-Jan Boons. Binding and Cellular Activation Studies Reveal That Toll-like Receptor 2 Can Differentially Recognize Peptidoglycan from Gram-positive and Gram-negative Bacteria. J Biol Chem. 2009 March 27; 284（13）: 8643–8653.
5. Jian-Wen Dong, Jesus G. Vallejo, Huei-Ping Tzeng, James A Thomas, Douglas L. Mann. Innate immunity mediates myocardial preconditioning through Toll-like receptor 2 and TIRAP-dependent signaling pathways. Am J Physiol Heart Circ Physiol. 2010 March; 298（3）: H1079–H1087.
6. Alexandre Jacques, Christian Bleau, Claire Turbide, Nicole Beauchemin, Lucie Lamontagne. Macrophage interleukin-6 and tumour necrosis factor-α are induced by coronavirus fixation to Toll-like receptor 2/heparan sulphate receptors but not carcinoembryonic cell adhesion antigen 1a. Immunology. 2009 September; 128（1pt2）: e181–e192.
訂購信息：
| 貨號 | 名稱 | 產地 | 規格 | 報價/元 | 貨期 |
| IML-202 | TLR2 Stable Cell Line | imgenex | 1Vial | 13328 | 2-3周 |
