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Glo luciferase assay system

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The Glo Luciferase Assay System is a sensitive luminescent reporter assay developed by Promega. It is designed to quantify the activity of firefly luciferase, a bioluminescent enzyme, in cell-based assays. The system provides reagents and protocols for the detection and measurement of luciferase activity.

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Lab products found in correlation

β galactosidase enzyme assay system, by Promega (2 mentions) Transfection reagent, by Roche (2 mentions) Dual luminescence assay kit, by GeneCopoeia (2 mentions) Psicheck 2, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Atdc5 cells, by Merck Group (1 mentions) Glo lysis buffer, by Promega (1 mentions) Ivis spectrum ct, by PerkinElmer (1 mentions) Glomax 20 20 luminometer, by Promega (1 mentions)

7 protocols using glo luciferase assay system

1

Luciferase Assay for IKK Signaling

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IKK wild-type and IKK mutant reporter vectors were constructed and inserted into the psiCHECK-2 (Promega, U.S.A.) [25 (link)]. The reporter plasmids (IKK wild-type or IKK mutant) together with si-GAS5 or si-control were transfected into SV40 MES-13 cells using Lipofectamine 2000 (catalog number: 11668019, Invitrogen) for 48 h for Luciferase reporter assay. Cells were collected to measure luciferase activity by dual Glo™ Luciferase Assay System (catalog number: E1910, Promega, U.S.A.).
Zhang R., Han X., Huang T, & Wang X. (2019). Danggui buxue tang inhibited mesangial cell proliferation and extracellular matrix accumulation through GAS5/NF-κB pathway. Bioscience Reports, 39(10), BSR20181740.
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2

Runx1 Regulation of Indian Hedgehog Promoter

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Ihh promoter sequences were analyzed for putative Runx binding sites with PROMO3.0 (http://alggen.lsi.upc.es/) using version 8.3 of the TRANSFAC database. The promoter region (−) and (+) of the mouse Ihh gene was amplified by PCR from a murine Ihh BAC clone (CH29–567C21; CHORI). Primer sequences are available in Table S3. Then the promoter regions were inserted into the pGL3-basic vector to construct the pGL3-Ihh promoter fragments. ATDC5 cells (Sigma) were cultured in 24-well plates, were transfected with the DNA mixture containing different pGL3-Ihh construct (0.3μg) and β-GAL-expressing plasmids (0.06 μg), with or without Runx1 expressing vector (psport6-CMV-Runx1, 0.3μg) using a calcium phosphate co-precipitation method. Luciferase was detected using Glo Luciferase Assay System (Promega) as described [20 (link), 22 (link)]. The β-GAL activity of the cell lysates was analyzed using β-Galactosidase Enzyme Assay System (E2000; Promega). The level of luciferase activity was normalized to the level of β-GAL activity.
Tang C.Y., Chen W., Luo Y., Wu J., Zhang Y., McVicar A., McConnell M., Liu Y., Zhou H.D, & Li Y.P. (2020). Runx1 up-regulates chondrocyte to osteoblast lineage commitment and promotes bone formation by enhancing both chondrogenesis and osteogenesis. The Biochemical journal, 477(13), 2421-2438.
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3

Promoter-driven Luciferase Assay

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The promoter region (−) and (+) of the mouse Bmp7, Alk3 and Atf4 gene was amplified by PCR using Bmp7, Alk3 and Atf4 Bac clone (cat#CH29-27K23; CHORI). Primer sequences are available in S3 Table. Then the promoter regions were inserted into the pGL3-basic vector to construct the pGL3-Bmp7, Alk3 and Atf4 promoter vectors and respectively. The insertions of the constructs were confirmed by sequencing. C3H10T1/2 cells were cultured in 24-well plates, and were transiently transfected with a DNA mixture containing the pGL3- Bmp7, Alk3 and Atf4 construct respectively (0.3μg) and β-GAL-expressing plasmids (0.03 μg using Lipofectamine and Plus reagents. Luciferase was detected using Glo Luciferase Assay System (Promega) 48 h post transfection as described [15 (link)]. The β-GAL activity of the cell lysates was analyzed using β-Galactosidase Enzyme Assay System (E2000; Promega). The level of luciferase activity was normalized to the level of β-GAL activity.
Tang C.Y., Wu M., Zhao D., Edwards D., McVicar A., Luo Y., Zhu G., Wang Y., Zhou H.D., Chen W, & Li Y.P. (2021). Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways. PLoS Genetics, 17(1), e1009233.
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4

Dual Luciferase Assay for STAT3 Regulation

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For the dual luciferase assay, 5637 cells were plated in triplicate into 12-well plates and cotransfected with 1 μg of the reporter construct, 15 pmol of the STAT3 luciferase reporter vector, and BUB1 siRNA by using transfection reagent (Roche). Transfected cells were cultured, and 24 h later, supernatants were collected for the luciferase assay using a Dual Luminescence Assay Kit (GeneCopoeia, MD) according to the manufacturer’s instructions. The luciferase assay was performed with the Glo Luciferase Assay System (Promega).
Jiang N., Liao Y., Wang M., Wang Y., Wang K., Guo J., Wu P., Zhong B., Guo T, & Wu C. (2021). BUB1 drives the occurrence and development of bladder cancer by mediating the STAT3 signaling pathway. Journal of Experimental & Clinical Cancer Research : CR, 40, 378.
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5

Dual Luciferase Assay for YAP1, SOX2, and Nanog

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For the dual luciferase assay, LNCaP and C4-2 cells were plated into 12-well plates and co-transfected with 1 μg of the seap reporter gene construct and 15 pmol of YAP1 luciferase together with siRNA of other plasmids by using transfection reagent (Roche). Transfected cells were cultured and 24 h later the supernatants were collected for luciferase assay using Dual Luminescence assay kit (GeneCopoeia MD) according to the manufacturer’s instructions. To measure SOX2 and Nanog promoter driven luciferase activity, the Glo Luciferase Assay System (Promega) was used.
Jiang N., Ke B., Hjort-Jensen K., Iglesias-Gato D., Wang Z., Chang P., Zhao Y., Niu X., Wu T., Peng B., Jiang M., Li X., Shang Z., Wang Q., Chang C., Flores-Morales A, & Niu Y. (2017). YAP1 regulates prostate cancer stem cell-like characteristics to promote castration resistant growth. Oncotarget, 8(70), 115054-115067.
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6

In Vivo Bioluminescence Imaging

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Isoflurane anesthetized mice were intravenously injected with luciferin (100 μL of 30 mg/mL per 20 g mouse weight) and imaged on an IVIS Spectrum CT (PerkinElmer, Waltham, USA). Living Image 4.5 software was used for data analysis. For ex vivo detection of luciferase activity, the respective organs were homogenized in Glo Lysis Buffer (Promega, Fitchburg, USA) and bioluminescence activity was detected with a bright Glo Luciferase Assay System (Promega, Fitchburg, USA).
Koestner W., Spanier J., Klause T., Tegtmeyer P.K., Becker J., Herder V., Borst K., Todt D., Lienenklaus S., Gerhauser I., Detje C.N., Geffers R., Langereis M.A., Vondran F.W., Yuan Q., van Kuppeveld F.J., Ott M., Staeheli P., Steinmann E., Baumgärtner W., Wacker F, & Kalinke U. (2018). Interferon-beta expression and type I interferon receptor signaling of hepatocytes prevent hepatic necrosis and virus dissemination in Coxsackievirus B3-infected mice. PLoS Pathogens, 14(8), e1007235.
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7

Regulation of PRDM1 by miR-125b

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The interaction between PRDM1 (encoding PR domain zinc finger protein 1) and hsa-miR-125b-5p (miR-125b) was predicted using TargetScan software (version 7.1) (23 (link)). Position 166–172 of the PRDM1 3’ UTR was predicted to bind to miR-125b. The PRDM1 fragment containing the miR-125b binding sites was synthesized to generate wild-type (PRDM1–WT) or mutant-type (PRDM1–MUT). The reporter of fluorescence in the vector was encoded by the Renilla Luciferase gene (Rluc), and PRDM1–WT and PRDM1–MUT were cloned downstream of Rluc the gene, separately. Then, the luciferase reporter plasmids PRDM1–WT and PRDM1–MUT (RiboBio, Guangzhou, China) were co-transfected with miR-125b mimic or control mimics (RiboBio) into HEK-293 T cells. Forty-eight hours after transfection, the Glo® Luciferase Assay System (Promega, Madison, WI, USA) was used to lyse the cells and a GLomax20/20 Luminometer (Promega) was used to determine the luciferase activity.
Xing Y., Li B., He J, & Hua H. (2022). Labial Gland Mesenchymal Stem Cell Derived Exosomes-Mediated miRNA-125b Attenuates Experimental Sjogren’s Syndrome by Targeting PRDM1 and Suppressing Plasma Cells. Frontiers in Immunology, 13, 871096.
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