FliC activity was characterized by a TLR-5-dependent luciferase activation assay in vitro. Hela cells (ATCC, Manassas, VA) were grown in DMEM supplemented with 10% fetal bovine serum (FBS), and cultured in humidified 5% CO2 at 37°C. Cells were incubated overnight at a density of 2*106 cells/well in a 6-well plate, and transfected the following day with 10 μg pUNO1-hTLR5, 2 μg pGL4.32-[Luc2/Nf-κB/Hygro] (Invivogen, San Diego, CA) and 15 μL Lipofectamine 2000 (Invitrogen, Grand Island, NY) in DMEM with 1% FBS. Transfected cells were plated the following day at a density of 5*104 cells/well in a 96-well plate in DMEM with 1% FBS. 1 μg/mL nanoparticles were suspended in fresh, DMEM + 1% FBS and used to stimulate transfected cells for 8 hours. Bright-Glo Luciferase Assay reagent (Promega, Madison, WI) was diluted 1:1 with serum-free DMEM and used to assess luciferase activity according to the manufacturer’s instructions.
Pgl4.32 luc2 nf κb hygro
Manufactured by InvivoGen
PGL4.32-[Luc2/Nf-κB/Hygro] is a reporter plasmid that contains the Firefly luciferase (Luc2) gene under the control of an NF-kB response element. It also includes a hygromycin resistance gene for selection purposes.
Automatically generated - may contain errors
2 protocols using pgl4.32 luc2 nf κb hygro
1
TLR-5-Dependent FliC Activity Assay
2
FliC Stimulation Activates NF-κB Pathway
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FliC activity was characterized by a TLR‐5‐dependent luciferase activation assay in vitro. Hela cells (ATCC, Manassas, VA) were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and cultured in humidified 5% CO2 at 37°C. Cells were incubated overnight at a density of 2 × 106 cells/well in a 6‐well plate, and transfected the following day with 10 μg pUNO1‐hTLR5, 2 μg pGL4.32‐[Luc2/Nf‐κB/Hygro] (Invivogen, San Diego, CA) and 15 μl Lipofectamine 2000 (Invitrogen, Grand Island, NY) in DMEM with 1% FBS. Transfected cells were plated the following day at a density of 5 × 104 cells/well in a 96‐well plate in DMEM with 1% FBS. Nanoparticles were suspended in fresh DMEM + 1% FBS at a concentration of 1 μg/mL and used to stimulate transfected cells for 8 hr. Bright‐Glo Luciferase Assay reagent (Promega, Madison, WI) was diluted 1:1 with serum‐free DMEM and used to assess luciferase activity according to the manufacturer's instructions.
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