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CYTOKINE SIGNALING 4
B16-Blue IFN-a/b - Murine Type I IFNs Reporter Cells
➥ Monitor the JAK/STAT pathway
➥ Monitor the RIG-I/MDA-5 pathway
➥ Detection of murine IFN-a and IFN-b
Background
Interferon-alpha (IFN-a) and interferon beta (IFN-b), play an important
role in viral infections. They bind to an IFN receptor complex consisting of
two alpha chains (IFNAR1 and IFNAR2) and recruit JAK1 and TyK2. These
kinases phosphorylate STAT1 and STAT2 leading to the formation of the
ISGF3 complex. ISGF3 binds to IFN-stimulated response elements (ISRE)
in the promoters of IFN-stimulated genes (ISG) to regulate their
expression (figure 1). IFN-a and IFN-b are produced in response to viral
pathogen associated molecular patterns (PAMPs), such as viral RNA and
DNA. These PAMPs are recognized by cytoplasmic pattern recognition
receptors including the RNA helicases RIG-I or MDA5. Stimulation of
B16-Blue IFN-a/b cells with RIG-I/MDA-5 agonists, such as transfected
poly(I:C), triggers the secretion of IFN-a and IFN-b.
Description
B16-Blue IFN-a/b cells allow the detection of bioactive murine type I IFNs
by monitoring the activation of the JAK/STAT/ISGF3 pathway. They derive
from the murine B16 melanoma cell line of C57B1/6 origin after stable
transfection with a SEAP reporter gene under the control of the IFN-a/binducible
ISG54 promoter.
Stimulation of B16-Blue IFN-a/b cells with murine IFN-a or IFN-b, or
type I IFN inducers, such as tranfected poly(I:C) or poly(dA:dT), activates
the JAK/STAT/ISGF3 pathway and triggers the subsequent production of
SEAP (figures 2&3). Levels of SEAP in the supernatant can be easily
determined with QUANTI-Blue , a medium that turns purple/blue in the
presence of SEAP and by reading the OD at 655 nm.
• Detection range for mIFN-a: 10 2 - 10 4 IU/ml
• Detection range for mIFN-b: 10 2 - 10 4 IU/ml
B16-Blue IFN-a/b cells are resistant to Zeocin
Contents and Storage
B16-Blue IFN-a/b cells are grown in RPMI medium, 2 mM L-glutamine,
10% FBS supplemented with 100 μg/ml Zeocin . Each vial contains
5-7x 10 6
cells and is supplied with 10 mg Zeocin , 50 mg Normocin and
1 pouch of QUANTI-Blue . Cells are shipped on dry ice.
PRODUCT QUANTITY CAT. CODE
B16-Blue IFN-a/b cells 5-7 x 10 6 cells bb-ifnab
Related Products
Blasticidin, page 17 Anti-hIFN-a-IgA , page 104
Zeocin , page 18 QUANTI-Blue page 14
Poly(I:C) (HMW), page 77 Poly(I:C) (HMW)/LyoVec , page 80
Poly(dA:dT) Naked, page 80 Poly(dA:dT)/LyoVec, page 80
106 www.invivogen.com/cytokine-sensor-cells
Figure 1: JAK/STAT and RIG-I/MDA-5 signaling pathways
Figure 2: Stimulation of B16-Blue IFN-a/b cells by recombinant human and
murine IFN-a and IFN-b was assessed by measuring the levels of SEAP using
QUANTI-Blue .
Figure 3: Response of B16-Blue IFN-a/b cells following their stimulation with
naked poly(I:C) or poly(:C)/LyoVec. Induction of type I IFNs was assessed by
determining the levels of SEAP using QUANTI-Blue .