Poly(dA:dT)/LyoVec
TM
Double-stranded B DNA complexed with LyoVec™
Catalog # tlrl-patc
For research use only
Version # 11H09-MM
Product information
mEtHodS
content:
• 4 x 25 µg lyophilized poly(dA:dT)/LyoVec™
Note: Each vial contains 25 µg of poly(dA-dT)•poly(dT-dA) complexed
with 50 µg LyoVec.
• 10 ml sterile endotoxin-free water
Preparation of stock solution (50 mg/ml)
- Add 500 ml sterile endotoxin-free water (provided) per vial of
poly(dA:dT)/LyoVec™. Mix gently. Allow at least 15 minutes for
complete solubilization.
Endotoxin level: <0.001 EU/mg
Storage:
- Poly(dA:dT)/LyoVec™ is provided lyophilized and shipped at room
temperature. Store lyophilized product at -20˚C for up to 12 months .
- Upon resuspension, store poly(dA:dT)/LyoVec™ at 4°C. Resuspended
product is stable 1 week when properly stored.
dEScriPtion
Poly(dA:dT) is a poly(deoxyadenylic-deoxythymidylic) acid sodium
salt. Poly(dA:dT) is a repetitive double-stranded DNA (dsDNA) sequence
of poly(dA-dT)•poly(dT-dA) and a synthetic analog of B-DNA.
Poly(dA:dT) is complexed with the cationic lipid LyoVec™ to facilitate
its uptake.
Intracellular poly(dA:dT) is recognized by several cytosolic DNA
sensors, such as ZBP1/DAI and LRRFIP1, triggering an immune
response1-5. ZBP1/DAI and LRRFIP1 bind poly(dA:dT) inducing type I
IFN production via TBK1/IRF-3 and b-catenin pathways, respectively3,4.
Additionally, poly(dA:dT) is recognized by AIM2 triggering the
formation of an inflammasome and the subsequent secretion of IL-1b
and IL-185.
Furthermore, transfected poly(dA:dT) can be transcribed by RNA
polymerase III into dsRNA with a 5’-triphosphate moiety (5’pppdsRNA) which is a ligand for RIG-I6,7. Thus poly(dA:dT) is indirectly
sensed by RIG-I leading to type I IFN production through the adaptor
molecule IPS-1 and the TBK1/IRF3 pathway8.
1. ishii KJ, et al., 2006. A Toll-like receptor-independent antiviral response induced by doublestranded B-form DNA. Nat Immunol. 7(1):40-8. 2. ishii KJ. & akira S., 2006. Innate immune
recognition of, and regulation by, DNA Trends Immunol. 27(11):525-32. 3. takaoka a. et al.,
2007. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune
response. Nature. 448(7152):501-5. 4. Yang P. et al., 2010. The cytosolic nucleic acid sensor
LRRFIP1 mediates the production of type I interferon via a beta-catenin-dependent pathway.
Nat Immunol. 11(6):487-94. 5. Jones JW. et al., 2010. Absent in melanoma 2 is required for
innate immune recognition of Francisella tularensis. PNAS, 107(21):9771-6. 6. ablasser a. et
al., 2009. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA
polymerase III-transcribed RNA intermediate. Nat Immunol. 10(10):1065-72. 7. chiu YH. et
al., 2009. RNA polymerase III detects cytosolic DNA and induces type I interferons through the
RIG-I pathway. Cell. 138(3):576-91. 8. takeshita f. & ishii KJ., 2008. Intracellular DNA
sensors in immunity. Curr Opin Immunol. 20(4):383-8.
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
Europe: +33 562-71-69-39
E-mail: info@invivogen.com
Website: www.invivogen.com
induction of type i ifns
Induction of type I IFNs with poly(dA:dT) can be studied in a variety
of cells including immortalized murine embryonic fibroblasts (MEFs),
the murine B16 melanoma cell line and HEK293 cells.
- Stimulate cells with 10 ng/ml - 10 mg/ml poly(dA:dT)/LyoVec™ for 18-24h.
- Monitor induction of type I IFNs by measuring the levels of IFN-a
and/or IFN-ß produced in the cell culture supernatants by ELISA or by
using InvivoGen’s SEAP reporter cells. InvivoGen provides
HEK-Blue™ IFN-a/b cells which detect human type I IFNs, and
B16-Blue™ IFN-a/b cells which detect murine type I IFNs.
induction of iL-1b in tHP-1 cells
THP-1 cells are grown in RPMI 1640 medium supplemented with 10%
heat inactivated fetal bovine serum, 2 mM L-glutamine and
antibacterial antibiotics such as penicillin/streptomycin or Normocin™.
THP-1 cells are grown in suspension to a density of 1.0x106 cells/ml in
tissue culture flasks.
1- Treat THP-1 cells with 0.5 mM (300 ng/ml) PMA for 3h at 37°C
in 5% CO2.
Note: PMA treatment increases the phagocytic properties of these cells
and induces the production of pro-IL-1b.
2- Wash cells gently with PBS and add fresh culture medium.
3- After 1 to 3 days, wash cells with PBS and add fresh culture medium.
4- Add 1 to 5 mg/ml poly(dA:dT)/LyoVec™.
5- Incubate from 6 hours to overnight at 37°C in 5% CO2.
Note: The production of pro-IL-1b can be further increased by priming
PMA-activated THP-1 cells with LPS.
6-The next day, detect mature IL-1b in the supernatant of poly(dA:dT)activated THP-1 cells by Western blot, ELISA or using HEK-Blue IL-1b
cells. Theses cells are specifically engineered to detect bioactive IL-1b.
rELatEd ProductS
Product
Poly(dA:dT) naked
Poly(dG:dC)/LyoVec™
Poly(dG:dC) naked
5’ppp-dsRNA
B16-Blue™ IFNa/b
HEK-Blue™ IFNa/b
HEK-Blue™ IL-1b
catalog code
tlrl-patc
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bb-ifnab
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3950 Sorrento Valley Blvd. Suite 100
San Diego, CA 92121 - USA