HEK293 cells were plated equally (10,000 cells per well) in 96-well white-walled plates. The wells were subjected to amino acid starvation conditions for various time points (0, 0.5, 1, 2, and 3 h, respectively). Proteasome-Glo Cell-Based Reagent (Promega, catalog # G8660) was prepared as per manufacturer's protocol and an equal volume was added to each well. The content of the plate was mixed at 700 RPM for 2 min and then incubated at room temperature for 10 min. Luminescence was read using a Synergy 2 Luminescence Plate Reader (Biotek).
Synergy 2 luminescence plate reader
Manufactured by Agilent Technologies
The Synergy 2 luminescence plate reader is a multi-mode detection instrument designed for a variety of luminescence-based assays. It provides sensitive and accurate measurements of luminescent signals in 96- or 384-well microplates.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Proteasome glo cell based reagent, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Pirespuro3 vector, by Takara Bio (1 mentions)
Fugene 6, by Promega (1 mentions)
Plvx ires mcherry vector, by Takara Bio (1 mentions)
Secrete pair gaussia luciferase assay kit, by GeneCopoeia (1 mentions)
5 protocols using synergy 2 luminescence plate reader
1
Proteasome Activity Assay in HEK293 Cells
2
IFNβ Promoter Luciferase Assay
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Cells were cotransfected with a plasmid containing a firefly luciferase gene under the control of an IFNβ promoter (p125-luc) and a plasmid containing a renilla luciferase gene (pRL-null) at a ratio of 30:1. Luciferase was quantified using the Dual Luciferase Reporter assay system (Promega) and a Synergy 2 luminescence plate reader (Bio-Tek).
3
Luciferase Reporter Assay for Amino Acid Starvation
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Cells were transfected with the luciferase reporter for 24 h followed by splitting equally in two wells of a six-well plate for another 24 h incubation. The cells were then subjected to either complete (control) or amino acid depleted media (starvation) for 1 h. Cell pellets were lysed in reporter lysis buffer (Promega) followed by centrifugation to clear the lysates. Luminescence reactions were initiated with Promega DLR (100 μL; Promega) added to the lysates (30 μL). Luciferase activities were measured using a Synergy 2 Luminescence Plate Reader (Biotek).
4
MECP2 Transcriptional Regulation Assay
5
Cbln1 Promoter Luciferase Assay
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The Cbln1 promoter luciferase construct was purchased from GeneCopoeia, Inc. (Cat# MPRM22597-PG02), which encodes Gaussia luciferase driven by a 1331 bp genomic fragment upstream of the coding sequence of the murine Cbln1 gene (Accession: NC_000074.6). The Ube3a expression construct was generated by amplifying the coding sequence of human Ube3a isoform III from Plasmid #37605 (Addgene) with primers 5’-TCTTCCACTAGTGCCACCATGGCCACAGCTTGTAAAAGATC-3’ and 5’-TCTTCCGGATCCTTACAGCATGCCAAATCCTTTGG-3’ and subcloning into the SpeI and BamHI sites of the pLVX-IRES-mCherry vector (Clontech Cat#631237). HEK293T cells were transfected in 96-well using Lipofectamine 3000 Reagent (ThermoFisher) with 50 ng Cbln1 promoter-Gluc plasmid and 50 ng of either pLVX-IRES-mCherry or pLVX-Ube3a-IRES-mCherry per well (n = 6 each condition). 48 hours after transfection, Gaussia luciferase activity was developed using the Secrete-Pair Gaussia Luciferase Assay Kit (Genecopoeia, Inc., Cat#LF061) and measured with the BioTek Synergy 2 luminescence plate reader.
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