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TLR3 Stable Cell Line、TLR3 穩(wěn)轉(zhuǎn)細(xì)胞株
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TLR3 Stable Cell Line
Description（描述）
The TLR3 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 3 （TLR3） with an N-terminal HA tag. TLR3 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 using the NF-kB/SEAPorter™ Assay Kit （IMK-515, Fig. 3）.
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 TLR3 stable line is blasticidin.
Note: The selection agent for the TLR3 stable line is blasticidin.
Application（應(yīng)用）
The TLR3 stable cell line can be used for TLR3 flow cytometric calibration and detection control as well as TLR3-dependent functional assays.
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 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 TLR3 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 TLR3 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 TLR3 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 TLR3 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 TLR3 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 TLR3 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(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 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.
TLR3 Stable Cell Line 1
Figure 1. Western blot analysis of TLR3 expression in the TLR3 stable cell line using an HA antibody （20 ug total protein/lane）. Legend. Vect: Vector control stable cell line （IML-200）; TLR3: TLR3 stable cell line （IML-203）.
Figure 2. Flow analysis of TLR3 expression in the TLR3 stable cell line. Intracellular expression of TLR3 in the TLR3 stable cell line was analyzed by flow cytometry using a FITC-conjugated TLR3 antibody （IMG-315C） and compared with the Vector control stable cell line （IML-200）. IMGENEX’s Intracellular TLR Staining Flow Kit （10098K） was used for this test.
Reference（參考文獻(xiàn)）
1. Jennifer E. Cole, Tina J. Navin, Amanda J. Cross, Michael E. Goddard, Lena Alexopoulou, Anuja T. Mitra, Alun H. Davies, Richard A. Flavell, Marc Feldmann, Claudia Monaco. Unexpected protective role for Toll-like receptor 3 in the arterial wall. Proc Natl Acad Sci U S A. 2011 February 8; 108（6）: 2372–2377.
2. Michela Deleidi, Penelope J. Hallett, James B. Koprich, Chee-Yeun Chung, Ole Isacson. The Toll-like receptor-3 agonist poly（I:C） triggers nigrostriatal dopaminergic degeneration. J Neurosci. 2010 December 1; 30（48）: 16091–16101.
3. Su-Jin Moon, Mi-Kyung Park, Hye-Jwa Oh, Seon-Yeong Lee, Seung-Ki Kwok, Mi-La Cho, Ji Hyeon Ju, Kyung-Su Park, Ho-Youn Kim, Sung-Hwan Park. Engagement of Toll-Like Receptor 3 Induces Vascular Endothelial Growth Factor and Interleukin-8 in Human Rheumatoid Synovial Fibroblasts. Korean J Intern Med. 2010 December; 25（4）: 429–435.
4. Yu Zhou, Xu Wang, Manqing Liu, Quan Hu, Li Song, Li Ye, Dunjin Zhou, Wenzhe Ho. A critical function of toll-like receptor-3 in the induction of anti-human immunodeficiency virus activities in macrophages. Immunology. 2010 September; 131（1）: 40–49.
5. Hideo Negishi, Tomoko Osawa, Kentaro Ogami, Xinshou Ouyang, Shinya Sakaguchi, Ryuji Koshiba, Hideyuki Yanai, Yoshinori Seko, Hiroshi Shitara, Keith Bishop, Hiromichi Yonekawa, Tomohiko Tamura, Tsuneyasu Kaisho, Choji Taya, Tadatsugu Taniguchi, Kenya Honda. A critical link between Toll-like receptor 3 and type II interferon signaling pathways in antiviral innate immunity. Proc Natl Acad Sci U S A. 2008 December 23; 105（51）: 20446–20451.
1. Jennifer E. Cole, Tina J. Navin, Amanda J. Cross, Michael E. Goddard, Lena Alexopoulou, Anuja T. Mitra, Alun H. Davies, Richard A. Flavell, Marc Feldmann, Claudia Monaco. Unexpected protective role for Toll-like receptor 3 in the arterial wall. Proc Natl Acad Sci U S A. 2011 February 8; 108（6）: 2372–2377.
2. Michela Deleidi, Penelope J. Hallett, James B. Koprich, Chee-Yeun Chung, Ole Isacson. The Toll-like receptor-3 agonist poly（I:C） triggers nigrostriatal dopaminergic degeneration. J Neurosci. 2010 December 1; 30（48）: 16091–16101.
3. Su-Jin Moon, Mi-Kyung Park, Hye-Jwa Oh, Seon-Yeong Lee, Seung-Ki Kwok, Mi-La Cho, Ji Hyeon Ju, Kyung-Su Park, Ho-Youn Kim, Sung-Hwan Park. Engagement of Toll-Like Receptor 3 Induces Vascular Endothelial Growth Factor and Interleukin-8 in Human Rheumatoid Synovial Fibroblasts. Korean J Intern Med. 2010 December; 25（4）: 429–435.
4. Yu Zhou, Xu Wang, Manqing Liu, Quan Hu, Li Song, Li Ye, Dunjin Zhou, Wenzhe Ho. A critical function of toll-like receptor-3 in the induction of anti-human immunodeficiency virus activities in macrophages. Immunology. 2010 September; 131（1）: 40–49.
5. Hideo Negishi, Tomoko Osawa, Kentaro Ogami, Xinshou Ouyang, Shinya Sakaguchi, Ryuji Koshiba, Hideyuki Yanai, Yoshinori Seko, Hiroshi Shitara, Keith Bishop, Hiromichi Yonekawa, Tomohiko Tamura, Tsuneyasu Kaisho, Choji Taya, Tadatsugu Taniguchi, Kenya Honda. A critical link between Toll-like receptor 3 and type II interferon signaling pathways in antiviral innate immunity. Proc Natl Acad Sci U S A. 2008 December 23; 105（51）: 20446–20451.
訂購信息：
| 貨號(hào) | 名稱 | 產(chǎn)地 | 規(guī)格 | 報(bào)價(jià)/元 | 貨期 |
| IML-203 | TLR3 Stably Transfected HEK 293 Cells | imgenex | 1Vial | 13328 | 2-3周 |
