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TLR1 Stable Cell Line、TLR1 穩(wěn)轉(zhuǎn)細胞株
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產(chǎn)品介紹
TLR1 Stable Cell Line
Description（描述）
TLR1 stable cell line expresses full-length human Toll-like receptor 1 （TLR1） with an N-terminal HA tag. TLR1 expression has been validated by Western blotting （Fig. 1） and flow cytometry （Fig. 2）.
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.
Note: The selection agent for the TLR1 stable line is blasticidin.
Note: The selection agent for the TLR1 stable line is blasticidin.
Application（應(yīng)用）
The TLR1 stable cell line can be used for TLR1 flow cytometric calibration and detection control.
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 TLR1 stable 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 TLR1 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 TLR1 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 TLR1 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 TLR1 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 TLR1 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 TLR1 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 TLR1 stable line.
• Wash hands after handling the TLR1 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 TLR1 stable line.
• Wash hands after handling the TLR1 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 TLR1 expression in the TLR1 stable cell line using an HA antibody （20 ug total protein/lane）. Legend. Vect: Vector/HEK 293 （IML-200）; TLR1: TLR1 stable cell line （IML-201）.
Figure 2. Cell surface expression of TLR1 in the TLR1 stable cell line was analyzed by flow cytometry using Alexa Fluor® 488-conjugated TLR1 antibody （IMG-5012AF488） and compared with the Vector/HEK 293 cell line （IML-200）. IMGENEX’s rabbit IgG isotype control （20304AF488） and the Surface TLR Staining Flow Kit （10099K） were used for this test. *Note: An endogenous level of TLR1 is present in HEK 293 cells.
Reference（參考文獻）
1. Mark M. Wurfel, Anthony C. Gordon, Tarah D. Holden, Frank Radella, Jeanna Strout, Osamu Kajikawa, John T. Ruzinski, Gail Rona, R. Anthony Black, Seth Stratton, Gail P. Jarvik, Adeline M. Hajjar, Deborah A. Nickerson, Mark Rieder, Jonathan Sevransky, James P. Maloney, Marc Moss, Greg Martin, Carl Shanholtz, Joe G. N. Garcia, Li Gao, Roy Brower, Kathleen C. Barnes, Keith R. Walley, James A. Russell, Thomas R. Martin. Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis. Am J Respir Crit Care Med. 2008 October 1; 178（7）: 710–720.
2. Sunny H. Wong, Sailesh Gochhait, Dheeraj Malhotra, Fredrik H. Pettersson, Yik Y. Teo, Chiea C. Khor, Anna Rautanen, Stephen J. Chapman, Tara C. Mills, Amit Srivastava, Aleksey Rudko, Maxim B. Freidin, Valery P. Puzyrev, Shafat Ali, Shweta Aggarwal, Rupali Chopra, Belum S. N. Reddy, Vijay K. Garg, Suchismita Roy, Sarah Meisner, Sunil K. Hazra, Bibhuti Saha, Sian Floyd, Brendan J. Keating, Cecilia Kim, Benjamin P. Fair, Julian C. Knight, Philip C. Hill, Richard A. Adegbola, Hakon Hakonarson, Paul E. M. Fine, Ramasamy M. Pitchappan, Rameshwar N. K. Bamezai, Adrian V. S. Hill, Fredrik O. Vannberg. Leprosy and the Adaptation of Human Toll-Like Receptor 1. PLoS Pathog. 2010 July; 6（7）: e1000979.
3. Hooman Izadi, Amirreza T. Motameni, Tonya C. Bates, Elias R. Olivera, Vega Villar-Suarez, Ila Joshi, Renu Garg, Barbara A. Osborne, Roger J. Davis, Mercedes Rincón, Juan Anguita. c-Jun N-Terminal Kinase 1 Is Required for Toll-Like Receptor 1 Gene Expression in Macrophages. Infect Immun. 2007 October; 75（10）: 5027–5034.
4. Elisabeth Elass, Laëtitia Aubry, Maryse Masson, Agnès Denys, Yann Guérardel, Emmanuel Maes, Dominique Legrand, Joël Mazurier, Laurent Kremer. Mycobacterial Lipomannan Induces Matrix Metalloproteinase-9 Expression in Human Macrophagic Cells through a Toll-Like Receptor 1 （TLR1）/TLR2- and CD14-Dependent Mechanism. Infect Immun. 2005 October; 73（10）: 7064–7068.
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. Christina L. Lancioni, Qing Li, Jeremy J. Thomas, XueDong Ding, Bonnie Thiel, Michael G. Drage, Nicole D. Pecora, Assem G. Ziady, Samuel Shank, Clifford V. Harding, W. Henry Boom, Roxana E. Rojas. Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2. Infect Immun. 2011 February; 79（2）: 663–673.
2. Sunny H. Wong, Sailesh Gochhait, Dheeraj Malhotra, Fredrik H. Pettersson, Yik Y. Teo, Chiea C. Khor, Anna Rautanen, Stephen J. Chapman, Tara C. Mills, Amit Srivastava, Aleksey Rudko, Maxim B. Freidin, Valery P. Puzyrev, Shafat Ali, Shweta Aggarwal, Rupali Chopra, Belum S. N. Reddy, Vijay K. Garg, Suchismita Roy, Sarah Meisner, Sunil K. Hazra, Bibhuti Saha, Sian Floyd, Brendan J. Keating, Cecilia Kim, Benjamin P. Fair, Julian C. Knight, Philip C. Hill, Richard A. Adegbola, Hakon Hakonarson, Paul E. M. Fine, Ramasamy M. Pitchappan, Rameshwar N. K. Bamezai, Adrian V. S. Hill, Fredrik O. Vannberg. Leprosy and the Adaptation of Human Toll-Like Receptor 1. PLoS Pathog. 2010 July; 6（7）: e1000979.
3. Hooman Izadi, Amirreza T. Motameni, Tonya C. Bates, Elias R. Olivera, Vega Villar-Suarez, Ila Joshi, Renu Garg, Barbara A. Osborne, Roger J. Davis, Mercedes Rincón, Juan Anguita. c-Jun N-Terminal Kinase 1 Is Required for Toll-Like Receptor 1 Gene Expression in Macrophages. Infect Immun. 2007 October; 75（10）: 5027–5034.
4. Elisabeth Elass, Laëtitia Aubry, Maryse Masson, Agnès Denys, Yann Guérardel, Emmanuel Maes, Dominique Legrand, Joël Mazurier, Laurent Kremer. Mycobacterial Lipomannan Induces Matrix Metalloproteinase-9 Expression in Human Macrophagic Cells through a Toll-Like Receptor 1 （TLR1）/TLR2- and CD14-Dependent Mechanism. Infect Immun. 2005 October; 73（10）: 7064–7068.
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. Christina L. Lancioni, Qing Li, Jeremy J. Thomas, XueDong Ding, Bonnie Thiel, Michael G. Drage, Nicole D. Pecora, Assem G. Ziady, Samuel Shank, Clifford V. Harding, W. Henry Boom, Roxana E. Rojas. Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2. Infect Immun. 2011 February; 79（2）: 663–673.
訂購信息：
| 貨號 | 名稱 | 產(chǎn)地 | 規(guī)格 | 報價/元 | 貨期 |
| IML-201 | TLR1 Stable Cell Line | imgenex | 1Vial | 13328 | 2-3周 |
