TLR7 Stable Cell Line

Description（描述）

The TLR7 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 7 (TLR7) with an N-terminal HA tag. TLR7 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（完全培养基）

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 TLR7 stable cell line is blasticidin.

Application（应用）

The TLR7 stable cell line can be used for TLR7 flow cytometric calibration and detection control as well as TLR7-dependent functional assays.

Product Handling Protocol（产品处理协议）

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 TLR7 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 TLR7 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 TLR7 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（安全注意事项）

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.

Figure 1. Western blot analysis of TLR7 expression in the TLR7 stable cell line using an HA antibody (20 ug total protein/lane). Legend. Vect: Vector control stable cell line (IML-200); TLR7: TLR7 stable cell line (IML-207).

Figure 2. Flow analysis of TLR7 expression in the TLR7 stable cell line. Intracellular expression of TLR7 in the TLR7 stable cell line was analyzed by flow cytometry using a FITC-conjugated TLR7 antibody (IMG-581C) and compared with the Vector control stable cell line (IML-200). IMGENEX’s Intracellular TLR Staining Flow Kit (10098K) was used for this test.

Figure 3. Functional analysis of the TLR7 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 TLR7 stable cell line were transfected with NF-kB/SEAP reporter plasmid for 16 h. Cells were stimulated with Imidazoquinoline resiquimod R-848 (IMG-2208) for 24 h followed by SEAP assay.

Reference
1. Erin L. Lousberg, Kerrilyn R. Diener, Cara K. Fraser, Simon Phipps, Paul S. Foster, Weisan Chen, Satoshi Uematsu, Shizuo Akira, Sarah A. Robertson, Michael P. Brown, John D. Hayball. J Virol. 2011 April; 85(7): 3385–3396.
2. Yoshinori Kitagawa, Mayu Yamaguchi, Min Zhou, Takayuki Komatsu, Machiko Nishio, Tsuyoshi Sugiyama, Kenji Takeuchi, Masae Itoh, Bin Gotoh. A Tryptophan-Rich Motif in the Human Parainfluenza Virus Type 2 V Protein Is Critical for the Blockade of Toll-Like Receptor 7 (TLR7)- and TLR9-Dependent Signaling.J Virol. 2011 May; 85(9): 4606–4611.
3. Jongdae Lee, Christina C. N. Wu, Ki Jeong Lee, Tsung-Hsien Chuang, Kyoko Katakura, Yu-Tsueng Liu, Michael Chan, Rommel Tawatao, Michelle Chung, Carol Shen, Howard B. Cottam, Michael M. C. Lai, Eyal Raz, Dennis A. Carson. Activation of anti-hepatitis C virus responses via Toll-like receptor 7. Proc Natl Acad Sci U S A. 2006 February 7; 103(6): 1828–1833.
4. Elad H. Kaufman, Allison D. Fryer, David B. Jacoby. Toll-like receptor 7 agonists are potent and rapid bronchodilators in guinea pigs. J Allergy Clin Immunol. 2011 February; 127(2): 462–469.
5. Terrence Town, Fengwei Bai, Tian Wang, Amber T. Kaplan, Feng Qian, Ruth R. Montgomery, John F. Anderson, Richard A. Flavell, Erol Fikrig. Toll-like Receptor 7 Mitigates Lethal West Nile Encephalitis via Interleukin 23-Dependent Immune Cell Infiltration and Homing. Immunity. 2009 February 20; 30(2): 242–253.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-207	TLR7 Stably Transfected HEK 293 Cells	imgenex	1Vial	13328	2-3周