The reovirus genome was recovered from the purified virus particles using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Isolated reovirus genome was transfected to cells pretreated with the cathepsin inhibitors using Lipofectamine 2000 Transfection Reagent (Invitrogen) at 200 ng/mL. A RIG-I agonist 5′-triphosphate double-stranded RNA (5′-ppp dsRNA) was purchased from InvivoGen (San Diego, CA) and similarly transfected at 1 μg/mL. Total RNA was recovered 24 hrs after transfection, and subsequently qRT-PCR analysis was performed as described above.
5 triphosphate double stranded rna 5 ppp dsrna
Manufactured by InvivoGen
Sourced in United States
5'-triphosphate double-stranded RNA (5'-ppp dsRNA) is a synthetic RNA molecule that contains a 5'-triphosphate group and forms a double-stranded structure. It is a laboratory tool used in research settings.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Duoset elisa, by R&D Systems (1 mentions)
Poly 1 c hmw, by InvivoGen (1 mentions)
Fluorescently labelled secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Ultrapure lipopolysaccharide lps from e coli 0111 b4, by InvivoGen (1 mentions)
Fsl 1, by InvivoGen (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
M csf, by R&D Systems (1 mentions)
Lps escherichia coli 0111 b4, by Merck Group (1 mentions)
Polyinosinic polycytidylic acid poly 1 c, by Merck Group (1 mentions)
Primary human astrocytes, by Lonza (1 mentions)
Amyloid precursor protein, by Abcam (1 mentions)
Rig 1, by AnaSpec (1 mentions)
Genjet plus dna in vivo tranfection reagent, by SignaGen (1 mentions)
5 protocols using 5 triphosphate double stranded rna 5 ppp dsrna
1
Reovirus genome extraction and transfection
2
Innate Immune Stimulant Protocols
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Ultra pure Lipopolysaccharide (LPS) from E. coli 0111:B4, Lipoteichoic acid (LTA), Pam3CSK4, FSL-1, HKLM, polyI:C (HMW), FlTC-labelled polyI:C (HMW), R848, CpG and 5’ triphosphate double stranded RNA (5’ ppp-dsRNA) were purchased from InvivoGen (San Diego, USA), M-CSF, ELISA DuoSets and IFNβ antibodies were obtained from R&D Systems, (Abington, UK). Fluorescently labelled secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (PA, USA). hBD3 (GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK) was from Peptides International, and cys-ser hBD3 and cys-ser-TAMRA hBD3 were from Almac (Almac Group Ltd, Craigavon, UK).
3
Toll-like Receptor Activation in Cells
4
RIG-1 Signaling in Primary Human Astrocytes
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Human astrocytes were grown in culture as described in de Rivero Vaccari et al. in 2012
[10 (link)]. Primary human astrocytes (Lonza Basel, Switzerland) were grown in culture in complete Astrocyte Growth Medium (Lonza Basel, Switzerland) for seven days. RIG-1 signaling was stimulated with 5′ triphosphate double-stranded RNA (5′ppp dsRNA, Invivogen San Diego, CA, USA) as a specific ligand to stimulate RIG-1 signaling at different concentrations (2 and 4 μg/ml) for 18 hours. After stimulation, cells were harvested and immunoblotted for RIG-1 (Anaspec Fremont, CA, USA), phosphorylated IRF3 (Novus Biologicals Littleton, CO, USA), amyloid precursor protein (Abcam Cambridge, MA, USA) and amyloid-β (Epitomics Burlingame, CA, USA) expression as described.
[10 (link)]. Primary human astrocytes (Lonza Basel, Switzerland) were grown in culture in complete Astrocyte Growth Medium (Lonza Basel, Switzerland) for seven days. RIG-1 signaling was stimulated with 5′ triphosphate double-stranded RNA (5′ppp dsRNA, Invivogen San Diego, CA, USA) as a specific ligand to stimulate RIG-1 signaling at different concentrations (2 and 4 μg/ml) for 18 hours. After stimulation, cells were harvested and immunoblotted for RIG-1 (Anaspec Fremont, CA, USA), phosphorylated IRF3 (Novus Biologicals Littleton, CO, USA), amyloid precursor protein (Abcam Cambridge, MA, USA) and amyloid-β (Epitomics Burlingame, CA, USA) expression as described.
5
Synthetic RIG-I Agonist and Recombinant HA Protein
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