Jurkat/NFAT-Luc T cells were seeded 16 h earlier, followed by incubation with different concentrations of human IL-1Ra for an additional 2 h. T cell activation was induced by adding 1.5 µg/ml ionomycin (Sigma-Aldrich) and 1 µM phorbol ester (PMA) (Selleck Chemicals, Houston, TX, USA) for 4 h. Luciferase assay was performed following the manufacturer’s guideline. In brief, the detection reagent, QUANTI-LucTM Gold (InvivoGen) was added to the supernatant of ionomycin/PMA induced-Jurkat/NFAT-Luc T cells. Luciferase activities were measured by a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific, Waltham, MA, USA).
Quanti luctm gold
Manufactured by InvivoGen
QUANTI-LucTM Gold is a sensitive luciferase detection reagent for quantifying the activity of luciferase reporter genes in cell-based assays. It provides a rapid and simple method for measuring luciferase expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by InvivoGen (1 mentions)
Varioskan flash spectral scanning multimode reader, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by InvivoGen (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
2 protocols using quanti luctm gold
1
NFAT-Luc T Cell Activation Assay
2
EphA2-Mediated Internalization Assay
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The day before experiments, 10,000 A549-Dual or A549-Dual RIG-I KO cells were plated in a flat-bottomed transparent 96-well plate in 90 μL DMEM complete medium. Antibodies were diluted to 400 nM in Opti-MEM (Thermo Fisher Scientific, Ref. 31985062). All siRNAs were diluted to 400 nM in Opti-MEM containing LipofectamineTM RNAiMAX (Thermo Fisher Scientific, Ref. 13778075) for 5 minutes according to manufacturer’s protocol. Then, 10 μL of these solutions were added to cells.
For EphA2-dependent internalization assay, cells were pre-incubated with increasing concentration of the naked antibody for 1 hours in order to degrade surface-EphA2, then washed with DPBS 1X before adding the antibody-siRNA conjugates for 72 h.
Luciferase assay (Quanti-LucTM Gold, Invivogen, Ref. rep-qlcg5) was performed in a 96-well flat-bottomed white plate according to the manufacturer’s protocol.
SEAP assay (Quanti-BlueTM, invivogen, Ref. rep-qbs) was performed in a 96-well flat-bottomed transparent plate according to the manufacturer’s protocol.
WST-1 assay (Roche, Merck, Ref. 11644807001) was performed according to the manufacturers’ protocol.
For EphA2-dependent internalization assay, cells were pre-incubated with increasing concentration of the naked antibody for 1 hours in order to degrade surface-EphA2, then washed with DPBS 1X before adding the antibody-siRNA conjugates for 72 h.
Luciferase assay (Quanti-LucTM Gold, Invivogen, Ref. rep-qlcg5) was performed in a 96-well flat-bottomed white plate according to the manufacturer’s protocol.
SEAP assay (Quanti-BlueTM, invivogen, Ref. rep-qbs) was performed in a 96-well flat-bottomed transparent plate according to the manufacturer’s protocol.
WST-1 assay (Roche, Merck, Ref. 11644807001) was performed according to the manufacturers’ protocol.
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