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Lightswitch luciferase assay kit

Manufactured by Active Motif
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The LightSwitch Luciferase Assay Kit is a laboratory tool used to measure luciferase reporter gene activity in cells. It provides a reliable and sensitive method for quantifying gene expression levels.

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Lab products found in correlation

Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions) Id3 luminometer, by Molecular Devices (2 mentions) Cd47 lightswitch promoter reporter goclones rensp, by SwitchGear Genomics (2 mentions) Cypridina tk control, by SwitchGear Genomics (2 mentions) Ptk cluc, by SwitchGear Genomics (2 mentions) Pierce cypridina luciferase glow assay kit, by Thermo Fisher Scientific (2 mentions) Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (2 mentions) Dharmafect duo transfection reagent, by Horizon Discovery (2 mentions) Quick change mutagenesis 2 kit, by Qiagen (2 mentions) Clariostar plus reader, by BMG Labtech (2 mentions) Pcmvha hezh2, by Addgene (1 mentions) Pcmv6 entry, by OriGene (1 mentions) Lipofectamine ltx plus reagent, by Thermo Fisher Scientific (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Superfect, by Qiagen (1 mentions) Living image v 4, by PerkinElmer (1 mentions) Fluostar optima, by BMG Labtech (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Mir 138 5p mimic, by Qiagen (1 mentions) Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (1 mentions)

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Lightswitch

15 protocols using lightswitch luciferase assay kit

1

Transcriptional Regulation of PLD2 Promoter

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Luciferase reporter assays were performed using LightSwitch Luciferase Assay Kit from Active Motif (Cat. # 32031). The reporter vector contained the PLD2 promoter region and a downstream 3’-RenSP luciferase region. Cells were co-transfected as previously described with plasmid DNA for either of the 2 transcription factors (Slug or Snail) and the LightSwitch PLD2 promoter vector in a 96 well plate for 36 hours. LightSwitch Luciferase Assay Reagent was added to the wells and the signal was read on a luminometer. The signal for each sample was calculated and normalized to a negative control sample.
Ganesan R., Mallets E, & Gomez-Cambronero J. (2015). The transcription factors Slug (SNAI2) and Snail (SNAI1) regulate phospholipase D (PLD) promoter in opposite ways towards cancer cell invasion. Molecular Oncology, 10(5), 663-676.
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2

VEGF Promoter Activity Assay

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Cells (5×104 cells/well) were seeded in 24-well plate, 24 hours prior to transfection with mixture containing 1 µg VEGF promoter plasmid DNA (S721026; Active Motif, Carlsbad, CA, USA), 3 µL transfection reagent FuGENE® HD (Active Motif), and Opti-MEM. Promoter activity was quantified 24 hours post-transfection using the LightSwitch™ Luciferase Assay Kit (Active Motif).
Chiang K.C., Huang S.T., Wu R.C., Huang S.C., Yeh T.S., Chen M.H., Hsu J.T., Chen L.W., Kuo S.F., Chueh H.Y., Juang H.H., Hung S.I., Yeh C.N, & Pang J.H. (2019). Interferon α-inducible protein 27 is an oncogene and highly expressed in cholangiocarcinoma patients with poor survival. Cancer Management and Research, 11, 1893-1905.
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3

Characterizing ICAM1 Promoter Activity

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The LightSwitch Luciferase Assay Kit (#32031), GoClone human ICAM1 promoter reporter (S708388), LightSwitch positive control RPL10 promoter (#32006) and negative control LightSwitch random promoter (#32006) were purchased from Active Motif (Carlsbad, CA, USA) and used in experiments according to the manufacturer’s instructions.
Bailey K.M., Julian C.M., Klinghoffer A.N., Bernard H., Lucas P.C, & McAllister-Lucas L.M. (2019). EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis. Oncotarget, 10(36), 3385-3399.
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4

ZO-1 Promoter Activity Assay

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786-O and ACHN cells were co-transfected with a Renilla luciferase plasmid containing the promoter region of ZO-1 (Active Motif) and pCMVHA hEZH2 (Addgene) or pCMV6-Entry (Origene). Promoter activity was determined using a LightSwitch Luciferase Assay Kit (Active Motif) according to the manufacturer’s instructions. pCMVHA hEZH2 was a gift from Kristian Helin (Addgene plasmid # 24230; http://n2t.net/addgene:24230 ; RRID:Addgene_24230) [29 (link)].
Imai-Sumida M., Dasgupta P., Kulkarni P., Shiina M., Hashimoto Y., Shahryari V., Majid S., Tanaka Y., Dahiya R, & Yamamura S. (2020). Genistein represses HOTAIR/chromatin remodeling pathways to suppress kidney cancer. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 54(1), 53-70.
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5

Quantifying miRNA-Mediated Luciferase Reporter Activity

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HeLa cells were transfected for 4 h with Synthetic miRNA Target GoClone Reporters (harboring two fully complementary miRNA binding sequence of interest at 3′UTR of the luciferase gene; Active Motif) and Human pre-microRNA Expression Construct Lenti-miRNAs of interest (System Biosciences) using Lipofectamine LTX-Plus reagent (Life Technologies). Cells were seeded overnight prior to transfection with anti-miR for 24 h using Lipofectamine RNAiMAX (Life Technologies). Luciferase activity was measured using LightSwitch Luciferase assay kit (Active Motif). Raw luciferase data was background subtracted and normalized to untreated control. EC50 values were estimated using GraphPad Prism software v7.04.
Lee E.C., Valencia T., Allerson C., Schairer A., Flaten A., Yheskel M., Kersjes K., Li J., Gatto S., Takhar M., Lockton S., Pavlicek A., Kim M., Chu T., Soriano R., Davis S., Androsavich J.R., Sarwary S., Owen T., Kaplan J., Liu K., Jang G., Neben S., Bentley P., Wright T, & Patel V. (2019). Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease. Nature Communications, 10, 4148.
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6

Investigating miR-221 Regulation of JNK2 3'UTR

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JNK2 3’UTR reporter construct (LightSwitch™ 3’UTR GoClone®, Product code 32012, Active Motif Inc.), which expresses the luciferase reporter gene fused to the 3′UTR of JNK2, was transfected into HEK293 cells (ATCC) together with miR-221 mimic or mimic control (40 nM), or miR-221 inhibitor or inhibitor control (40 nM). At 48 h, luciferase activity was measured using LightSwitch™ Luciferase Assay Kit (Active Motif Inc.) according to the manufacturer’s instructions.
Yang J., Do-Umehara H.C., Zhang Q., Wang H., Hou C., Dong H., Perez E.A., Sala M.A., Anekalla K.R., Walter J.M., Liu S., Wunderink R.G., Budinger G.R, & Liu J. (2021). miR-221-5p-Mediated Downregulation of JNK2 Aggravates Acute Lung Injury. Frontiers in Immunology, 12, 700933.
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7

CD47 Promoter Reporter Assay

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The luciferase reporter assay was performed as previously described11 (link). CD47 LightSwitch Promoter Reporter GoClones (RenSP, S710450) and Cypridina TK Control constructs (pTK-Cluc, SN0322S) were obtained from SwitchGear Genomics. 45 ng of the RenSP reporter and 5 ng of the pTK-Cluc reporter construct were transfected into mouse smooth muscle cells using Lipofectamine 3000 Transfection Reagent (Thermo Scientific, Catalogue# L3000–008) and Opti-MEM I Reduced Serum Medium (Thermo Scientific, Catalogue# 31985062). After 48 hours, media was changed to fresh medium and cells were then exposed to DMSO, 50 ng/ml TNF-α + DMSO, or 50 ng/ml TNF-α + 10 μM atorvastatin. The cell lysate and supernatant were harvested 24 hours after stimulation/treatment und dual luciferase activity was measured with the LightSwitch Luciferase Assay Kit (Active Motif, Catalogue# 32031, NC0999256) and Pierce Cypridina Luciferase Glow Assay Kit (Thermo Scientific, PI16170) using an iD3 luminometer (Molecular Devices). Relative luciferase activity (RenSP/Cypridina ratio) was quantified as the percentage change relative to the basal value obtained from control-transfected cells.
Jarr K.U., Ye J., Kojima Y., Ye Z., Gao H., Schmid S., Luo L., Baylis R.A., Lotfi M., Lopez N., Eberhard A.V., Smith B.R., Weissman I.L., Maegdefessel L, & Leeper N.J. (2022). The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis. Nature cardiovascular research, 1(3), 253-262.
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8

PD-L1 Promoter Luciferase Assay

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Cells were transfected with 1.5 µg of PD-L1 promoter-luciferase reporter (pPD-L1-Luc) or control vector (Active Motif, Carlsbad, CA, USA) in the presence of Superfect (Qiagen, Germantown, MD, USA). After 48 h, the cells were lysed in passive lysis buffer. Lysates were analyzed using the Lightswitch Luciferase Assay Kit (Active Motif).
Maeda T., Hiraki M., Jin C., Rajabi H., Tagde A., Alam M., Bouillez A., Hu X., Suzuki Y., Miyo M., Hata T., Hinohara K, & Kufe D. (2017). MUC1-C INDUCES PD-L1 AND IMMUNE EVASION IN TRIPLE-NEGATIVE BREAST CANCER. Cancer research, 78(1), 205-215.
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9

Validating miRNA regulation of SOX9

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The pLS-SOX9 plasmid containing the SOX9 3′-UTR fused to the renilla luciferase reporter gene was purchased from Active motif (Active motif, La Hulpe, Belgium). In reporter assays, 100 ng pLS-vector were co-transfected with 50 nM miRNA using the DharmaFect Duo transfection reagent (GE Dharmacon, Lafayette, CO) according to the manufacturer’s protocol into cells seeded in 96-well cell culture plates on day prior to transfection. One day after transfection, luciferase signals were measured using the Lightswitch Luciferase Assay kit (Active Motif, La Hulpe, Belgium) according to the manufacturer´s protocol. Luminescence signal was assessed with CLARIOstar Plus reader (BMG LABTECH, Ortenberg, Germany). To verify direct binding, mutation of the miRNA binding site within the SOX9 3′-UTR was performed using the Quick-change mutagenesis II kit (Qiagen, Hilden, Germany) as outlined in the manufacturer’s protocol. Primers used for mutagenesis are listed in Table S1.
Chao T.Y., Kordaß T., Osen W, & Eichmüller S.B. (2022). SOX9 is a target of miR-134-3p and miR-224-3p in breast cancer cell lines. Molecular and Cellular Biochemistry, 478(2), 305-315.
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10

CD47 Promoter Reporter Assay

Cited 1 time
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The luciferase reporter assay was performed as previously described11 (link). CD47 LightSwitch Promoter Reporter GoClones (RenSP, S710450) and Cypridina TK Control constructs (pTK-Cluc, SN0322S) were obtained from SwitchGear Genomics. 45 ng of the RenSP reporter and 5 ng of the pTK-Cluc reporter construct were transfected into mouse smooth muscle cells using Lipofectamine 3000 Transfection Reagent (Thermo Scientific, Catalogue# L3000–008) and Opti-MEM I Reduced Serum Medium (Thermo Scientific, Catalogue# 31985062). After 48 hours, media was changed to fresh medium and cells were then exposed to DMSO, 50 ng/ml TNF-α + DMSO, or 50 ng/ml TNF-α + 10 μM atorvastatin. The cell lysate and supernatant were harvested 24 hours after stimulation/treatment und dual luciferase activity was measured with the LightSwitch Luciferase Assay Kit (Active Motif, Catalogue# 32031, NC0999256) and Pierce Cypridina Luciferase Glow Assay Kit (Thermo Scientific, PI16170) using an iD3 luminometer (Molecular Devices). Relative luciferase activity (RenSP/Cypridina ratio) was quantified as the percentage change relative to the basal value obtained from control-transfected cells.
Jarr K.U., Ye J., Kojima Y., Ye Z., Gao H., Schmid S., Luo L., Baylis R.A., Lotfi M., Lopez N., Eberhard A.V., Smith B.R., Weissman I.L., Maegdefessel L, & Leeper N.J. (2022). The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis. Nature cardiovascular research, 1(3), 253-262.
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