Poly(I:C) (HMW)高分子量 

BACKGROUND:

Polyinosinic-polycytidylic acid (poly(I:C)) is a synthetic analog of double stranded RNA (dsRNA), a molecular pattern associated with viral infection. Both natural and synthetic dsRNAs are known to induce type I interferons (IFN) and other cytokines production. Poly(I:C) is recognized by Toll-like receptor 3 (TLR3)1, 2. Upon poly(I:C) recognition, TLR3 activates the transcription factor interferon regulatory factor 3 (IRF3), through the adapter protein Toll-IL-1 receptor (TIR) domain containing adapter inducing IFN-β (TRIF, also known as TICAM-1)3. Activation of IRF3 leads to the production of type I IFNs, especially IFN-β. A second pathway involves the recruitment of TNF receptor associated factor 6 (TRAF6) or receptor interacting protein 1 (RIP1), with the subsequent activation of the transcription factors NF-κB and AP-14. Activation of this pathway triggers the production of inflammatory cytokines and chemokines such as TNF-α, IL-6 and CXCL10. Poly(I:C) is also recognized by the cytosolic RNA helicases retinoic acid-inducible protein I (RIG-I) and melanoma differentiation associate gene 5 (MDA-5)⁵.

PRODUCT DESCRIPTION:

Polyinosinic-polycytidylic acid (usually abbreviated as poly(I:C) or poly(rI):poly(rC)) is a synthetic analog of double-stranded RNA (dsRNA), a molecular pattern associated with viral infection. Poly(I:C) activates the antiviral pattern recognition receptors TLR3, RIG-I/MDA5 and PKR, thereby inducing signaling via multiple inflammatory pathways, including NF-κB and IRF. High Molecular Weight Poly(I:C) comprises long strands of inosine poly(I) homopolymer annealed to strands of cytidine poly(C) homopolymer. The average size of Poly(I:C) HMW is 1.5 to 8 kb.

Quality control:

- Absorbance spectrum

- TLR3 activity has been verified using HEK-Blue™ TLR3 cells.

- The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using cellular assays.

Specifications:

Storage and stability:

- Product is shipped at room temperature. Upon receipt, store at 4°C.

- Upon resuspension, prepare aliquots of Poly(I:C) HMW and store at 4°C or at -20°C. Resuspended product is stable for 1 month at 4°C and 1 year at -20°C. Avoid repeated freeze-thaw cycles.

METHODS:

Preparation of stock solution (1 mg/ml) Stimulation of TLR3 can be achieved with 30 ng - 10 µg/ml Poly(I:C).

- Add 10 ml of the endotoxin-free physiological water to the 10 mg Poly(I:C) vial or 50 ml to the 50 mg Poly(I:C) vial to obtain a solution at 1 mg/ml.

- Mix the solution by pipetting up and down.

- Heat the mixture for 10 minutes at 65-70°C. Allow the solution to cool for 1 hour at room temperature to ensure proper annealing.

TLR3 activation of TLR3 with Poly(I:C)HMW:

Poly(I:C) HMW can be used to stimulate hTLR3 in HEK-Blue™ hTLR3 cells. These cells are designed for studying the stimulation of hTLR3 by monitoring the activation of NF-κB. Stimulation with a TLR3 ligand activates NF-κB and AP-1 which induces the production of SEAP. Levels of SEAP can be easily determined with QUANTI-Blue™ Solution, a detection medium that turns purple/blue in the presence of alkaline phosphatase.