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Biolux cypridina luciferase assay kit

Manufactured by New England Biolabs
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The BioLux Cypridina Luciferase Assay Kit is a bioluminescent assay system designed for the quantitative detection of Cypridina luciferase activity. The kit includes Cypridina luciferase substrate, assay buffer, and standard solutions necessary for the assay.

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Lab products found in correlation

Renilla luciferase assay system, by Promega (2 mentions) Transit mrna transfection kit, by Mirus Bio (2 mentions) Lmaxii384, by Molecular Devices (1 mentions) Renilla luciferase assay reagent, by Promega (1 mentions) Lumat lb 9507, by Berthold Technologies (1 mentions) Dual reporter assay system, by Promega (1 mentions) Centro lb 960, by Berthold Technologies (1 mentions) Biolux gaussia luciferase flex assay kit, by New England Biolabs (1 mentions) Emerald luc luciferase assay reagent, by Toyobo (1 mentions) Infinite f500 luminometer, by Tecan (1 mentions) Centro lb 960 luminometer, by Berthold Technologies (1 mentions)

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BioLux luciferase assay kit

7 protocols using biolux cypridina luciferase assay kit

1

Quantifying Luciferase Reporter Activity

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Collected supernatants were immediately mixed with an equal amount of 1X lysis buffer. Twenty μl of lysed sample was loaded on to a 96-well plate in triplicate. Gaussia luciferase (GLuc) activity was detected using the Renilla Luciferase Assay System (Promega, USA). Relative light units (RLUs) were measured with a spectramax plate reader (L MAXII 384, Molecular Devices, USA). Luminescence was integrated over 10 sec with a 2-second delay and measured upon injection of 50 μl of substrate reagent. For Cypridina luciferase (normalizer) measurement, we used BioLux Cypridina Luciferase Assay kit (NEB, USA) with identical settings to GLuc measurement except we set the integration time to 2 sec.
Nawtaisong P., Fraser M.E., Carter J.R, & Fraser MJ J.r. (2015). Trans-splicing Group I Intron Targeting Hepatitis C Virus IRES Mediates Cell Death upon Viral Infection in Huh7.5 Cells. Virology, 481, 223-234.
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2

Dual Luciferase Activity Assay

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10–50 ulof culture medium were used to measure GLuc (Renilla Luciferase Assay System, Promega) and CLuc (BioLux® Cypridina Luciferase Assay Kit, NEB). The relative GLuc activity was normalized to CLuc.
Li F., Cheng L., Murphy C.M., Reszka-Blanco N.J., Wu Y., Chi L., Hu J, & Su L. (2016). Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs. Scientific Reports, 6, 36483.
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3

Quantifying HBV cccDNA Activity with mcHBV-Gluc

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The Gluc signal from mcHBV-Gluc arises from pgRNA, providing a surrogate for cccDNA activity, as it is secreted from cells into the media. Gluc activity in aliquots of media was measured using the reagent for measurement of Renilla luciferase activity in the Dual Reporter Assay system (Promega) because Gaussia and Renilla catalyze their light-producing reactions using the same substrate. To measure Gluc, 10 μL of culture medium was added to 50 μL Renilla luciferase assay reagent (Promega), and luminescence was measured with a luminometer (Lumat LB 9507; Berthold Technologies, Bad Wildbad, Germany) with a 10-second integration. For Cluc, 1.0 μL of culture medium was added to 10 μL Cluc assay solution (BioLux Cypridina Luciferase Assay Kit, New England BioLabs), and luminescence was measured. The relative Gluc activity was normalized to Cluc unless otherwise specified. When using the endpoint mcHBV DNA copy number for normalization, the quantitation of mcHBV-Gluc DNA was conducted through qPCR using primers for the Gluc lesion as follows: 5′-ATGGTGAATGGCGTGAAG-3′ (sense) and 5′-TAGGTGTCATCGCCGCCAGC-3′ (antisense).34 (link)
Sekiba K., Otsuka M., Ohno M., Yamagami M., Kishikawa T., Suzuki T., Ishibashi R., Seimiya T., Tanaka E, & Koike K. (2018). Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx–DDB1 Interaction. Cellular and Molecular Gastroenterology and Hepatology, 7(2), 297-312.
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4

Measuring Transfected CLuc mRNA Expression

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Synthesized, purified mRNAs were transfected into human embryonic kidney cells using the TransIT-mRNA Transfection Kit as recommended by the manufacturer (Mirus Bio). The expression of the CLuc mRNA was analyzed by measuring the luciferase activity from the media at various time points (15, 30, 60, 90, 120, 180, 240, 300, 360 min post-transfection). The luciferase activity was measured with BioLux Cypridina Luciferase Assay Kit (New England Biolabs) using a Centro LB 960 luminometer (Berthold) in relative light units (RLU).
Wu M.Z., Asahara H., Tzertzinis G, & Roy B. (2020). Synthesis of low immunogenicity RNA with high-temperature in vitro transcription. RNA, 26(3), 345-360.
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5

Quantifying Secreted and Intracellular Luciferase

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CHO cells carrying the MEEVS‐reporter‐MAC2 were plated at a density of 2 × 105 in 24‐well tissue culture plates and cultured for 48 h. The conditioned media and cells were separately collected and used to measure luciferase activity. Activities of GLuc and CLuc luciferase secreted into the supernatant were measured using a BioLux® Gaussia Luciferase Flex Assay Kit (NEB) and a BioLux® Cypridina Luciferase Assay Kit (NEB), respectively. Twenty microliter of various conditioned cell culture media were transferred to flat solid bottom and opaque‐walled white 96‐well plates (Nunc, Roskilde, Denmark). GLuc and CLuc bioluminescence values were measured immediately after the addition of 50 μL of substrate solution. Expression of ELuc in cells was measured with the Emerald Luc Luciferase Assay Reagent (TOYOBO). Cells were resuspended at a density of 1 × 105 cells per 50 μL with PBS and transferred to flat solid bottom and opaque‐walled white 96‐well plates (Nunc). ELuc bioluminescence values were measured after the addition of 50 μL of assay reagent and incubation for 10 min. An Infinite F500 luminometer (Tecan, Crailsheim, Germany) was used to determine luciferase activity.
Tomimatsu K., Kokura K., Nishida T., Yoshimura Y., Kazuki Y., Narita M., Oshimura M, & Ohbayashi T. (2017). Multiple expression cassette exchange via TP901‐1, R4, and Bxb1 integrase systems on a mouse artificial chromosome. FEBS Open Bio, 7(3), 306-317.
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6

Luciferase Reporter Gene Assay Protocol

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Luciferase reporter gene assays were performed as previously described (8 (link)) with the exception that the BioLux Cypridina Luciferase Assay Kit (E3309L, New England Biolabs) was used. All assays were normalized to β-gal activity.
Calderon M.R., Verway M., Benslama R.O., Birlea M., Bouttier M., Dimitrov V., Mader S, & White J.H. (2014). Ligand-dependent corepressor contributes to transcriptional repression by C2H2 zinc-finger transcription factor ZBRK1 through association with KRAB-associated protein-1. Nucleic Acids Research, 42(11), 7012-7027.
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7

Quantifying mRNA Transfection Efficiency

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Synthesized, purified mRNAs were transfected into human embryonic kidney cells (HEK 293) using the TransIT-mRNA Transfection Kit (Mirus Bio). The expression from the luciferase mRNA was analyzed by measuring the luciferase activity from the media measured 6 h post-transfection. The luciferase activity was measured with BioLux Cypridina Luciferase Assay Kit (New England Biolabs) using a Centro LB 960 luminometer (Berthold) in relative light units.
MalagodaPathiranage K., Cavac E., Chen T.H., Roy B, & Martin C.T. (2023). High-salt transcription from enzymatically gapped promoters nets higher yields and purity of transcribed RNAs. Nucleic Acids Research, 51(6), e36.
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