PC12 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 5% FBS (ThermoFisher, USA) and 1% penicillin/streptomycin and were transfected with mimic124-3p/nc at a concentration of 50 nM oligonucleotides using Lipofectamine 3000 (Invitrogen), according to the manufacturer's protocol. For the luciferase reporter assay, pmirGLO dual-luciferase vectors (GenePharma, Shanghai, China) were used to construct dual-luciferase reporter plasmids. The sequences of miR-124-3p and circKcnk9 were separately cloned into vectors (Supplementary Figure S2 ). PC12 cells were co-transfected with wild-type pmirGLO-circKcnk9 or mutated type and miR-124 mimics (negative control). After 48 h induction, the luciferase activity was assessed using a dual-luciferase reporter kit (Promega, Madison, WI, USA). GloMax® 20/ 20 system (Promega, USA) was used to test luciferase activity. The relative firefly luciferase activity was normalized to Renilla luciferase activity.
Glomax 20 20 system
Manufactured by Promega
Sourced in United States
The GloMax 20/20 system is a luminometer designed for sensitive detection and quantification of luminescent signals. It provides accurate and reproducible measurements of various luciferase-based reporter assays.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay kit, by Promega (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pmirglo dual luciferase vector, by GenePharma (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter kit, by Promega (1 mentions)
Bactiter glo microbial cell viability assay kit, by Promega (1 mentions)
Pmirglo dual luciferase reporter, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Primestar dna polymerase, by Takara Bio (1 mentions)
9 protocols using glomax 20 20 system
1
Investigating miR-124-3p and circKcnk9 Interaction
2
Quantifying Microbial ATP via Luminometry
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A Luminometer (Glomax 20/20 System, Promega with BacTiter-Glo™ Microbial Cell Viability Assay kit) was used for estimating the total ATP of the living cells present in the enrichment samples (Adhikari and Kallmeyer, 2010 (link)). The details of the protocol were same as provided by the manufacturer, i.e the BacTiter-Glo™ Microbial Cell Viability Assay Technical Bulletin #TB337. Light emission was measured (as relative light units per second, RLU s−1) for three, five seconds intervals with a five seconds delay before each interval, which was then quantified as mM with reference to standard ATP curve. Sterile tips were used in transferring all solutions and samples to exclude ATP contamination of pipettes and solutions. All procedures were performed in triplicates in the dark and all plastic material, solutions, and pipettes were stored in the dark as light could hamper the results causing delayed fluorescence.
3
Pseudotyped Particle Infection Assay
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A total of 2.5 × 105 Huh-7, Vero-E6 and MRC-5 cells were seeded in 24-well plates and incubated at 37 °C for 24 h. The cells were washed with PBS, and 200 μL pseudotyped particles, corresponding to an average of 1.8 × 107 particles for all pseudovirion types as determined by nanoparticle tracking analysis (Supplementary Figure 2A ), were added to cells and incubated at 37 °C for 2 h. Complete medium was then added and cells were incubated at 37 °C for 72 h, after which luciferase activity was measured using Luciferase Assay Kit (Promega, Madison, WI, USA), and luminometer readings performed with a GloMax 20/20 system (Promega).
4
Luciferase Assay for miRNA-155-5p Target Validation
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Luciferase reporter gene assays were performed as previously described with some modifications.21 The online tool, TargetScanHuman,22 was used to predict possible miR‐155‐5p‐binding sites in SOCS1. Fragments of the wild‐type 3′ untranslated region (UTR) of SOCS1 containing the predicted target sites of miR‐155‐5p were cloned into the pmirGLO dual‐luciferase reporter (Promega Corporation,). Mutant constructs were generated with the target sites mutated, as specified in the “Results” section. Firefly luciferase was used as the primary reporter to investigate miRNA binding to the 3′‐UTR and subsequent regulation of gene expression. Renilla luciferase was used as an internal control for normalization. Human embryonic kidney 293T cells were seeded in 96‐well plates and co‐transfected with luciferase reporters (0.05 μg/well) and an miR‐155‐5p agomir or a SNC oligo (15 nM). Luciferase activities were measured 72 hours later using the Promega GloMax 20/20 system. Firefly luciferase activity was normalized to Renilla luciferase activity to assess the regulatory effect of miR‐155‐5p on its putative targets.
5
Curcumin Modulates Luciferase Expression
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After cotransfection with 100 ng pGL3.0-Qp and 5 ng pRL-TK for 4 h, HeLa cells were incubated with 0.006% DMSO or curcumin (5 or 10 μΜ). After 24 h, the total protein was harvested with the 1Χ lysis buffer (Promega, Madison, WI, USA). Luciferase activity was measured by the GLO-MAX 20/20 system (Promega, Madison, WI, USA). Renilla was as internal control reference.
6
Modulating Wnt Signaling Pathway
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We transfected HCT116, HT29 and DLD-1 cells with TOP or FOP flash plasmids along with SV40-Renilla plasmid (control for transfection) using Lipofectamine 3000 (Invitrogen). On the following day, we transferred the cells to fresh media and treated them with DMSO, 2 µM JIB-04, or 10 µM salinomycin. Forty-eight hours after transfection, we detected luciferase activity using the Promega GLOMAX 20/20 system and dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol.
7
SARS-CoV-2 Pseudotyped Virus Entry Assay
8
Extracellular Calcium Depletion Assay for SARSpp
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Vero-E6 cells (1.5 × 105) were seeded in 24-well plates and incubated at 37 °C 5% CO2 for 24 h. For extracellular calcium depletion assays, cells were washed twice with PBS with or without Ca2 +, and incubated with 200 μL of 2% FBS DMEM with or without Ca2 + for 2 h at 37 °C 5% CO2. Supernatants were removed and 200 μL of SARSpp solution with or without EDTA (1.5 mM) was added to the cells for 2 h at 37 °C 5% CO2. For experiments using BAPTA-AM intracellular calcium chelating reagent, cells were washed twice with PBS with or without Ca2 +, and incubated with a 200-μL solution of 2% FBS DMEM with either BAPTA-AM (50 μM) or DMSO for 2 h at 37 °C 5% CO2. Supernatants were removed and 200 μL of SARSpp solution containing either BAPTA-AM (50 μM) or DMSO was added to the cells for 2 h at 37 °C 5% CO2. For both types of assays, complete medium was then added and the cells were incubated at 37 °C for an additional 72 h. Luciferase activity was measured using Luciferase Assay Kit (Promega), and luminometer readings were performed with a GloMax 20/20 system (Promega).
9
Validating miRNA-mRNA Interactions
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Dual luciferase reporter assays were performed to verify the direct interactions between MCM3AP-AS1 and miR-15a as well as miR-15a and the 3′-UTR of EIF4E mRNA. PCR was conducted using the PrimeSTAR DNA polymerase (Takara) to amplify the MCM3AP-AS1 complementary DNA (cDNA) containing the predicted miR-15a binding site and the 3′-UTR of EIF4E cDNA containing the predicted miR-15a binding site. The products were purified and incorporated into the pmirGLO vector for further transfections. The pRL-TK plasmid was co-transfected as the internal control. After 48 hours of transfection, a luciferase assay kit (Promega, Madison, WI) and a Promega GloMax 20/20 system were used for final measurement.
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