Lumat lb 9501

Manufactured by Berthold Technologies

Sourced in Germany

The Lumat LB 9501 is a luminometer designed for the measurement of luminescence. It is capable of detecting and quantifying various forms of luminescence, including bioluminescence, chemiluminescence, and others. The Lumat LB 9501 provides reliable and accurate measurements of luminescence signals.

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Lab products found in correlation

Luciferase assay system, by Promega (7 mentions) Dual luciferase reporter assay system, by Promega (4 mentions) Lipofectamine, by Thermo Fisher Scientific (2 mentions) Passive lysis buffer (plb), by Promega (2 mentions) Atp determination kit a22066, by Thermo Fisher Scientific (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Adenosine assay kit, by Cell Biolabs (1 mentions) Luminol, by Merck Group (1 mentions) Luciferase assay system kit, by Promega (1 mentions) L 012 stock solution, by Fujifilm (1 mentions) Pg5luc, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Hela cell, by Thermo Fisher Scientific (1 mentions) Atp bioluminescence assay kit cls 2, by Roche (1 mentions) Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions) Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions) Superfect transfection reagent, by Qiagen (1 mentions) Glo lysis buffer, by Promega (1 mentions) Hiv 1 p24 elisa kit, by ZeptoMetrix (1 mentions) Fluostar omega, by BMG Labtech (1 mentions)

Spelling variants of this product

Lumat 9501 (3 citations) Lumat LB 9501/16 (3 citations) Lumat LB 9501 Single Tube Luminometer (2 citations)

39 protocols using lumat lb 9501

Astrocyte Metabolic Activity Assays

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ATP levels in the astrocyte supernatants and cells were measured using a luciferin/luciferase assay (ATP Determination Kit A22066; Invitrogen) and a luminometer (Berthold Lumat LB 9501) according to the manufacturer's instructions. Adenosine levels were measured on astrocytes supernatants using a fluorometric assay (Adenosine Assay Kit; Cell Biolabs, INC.) and a Fluorometer (TECAN) according to the manufacturer's instructions.
For determination of nucleotide hydrolysis free phosphate was measured using the Malachite Green Phosphate Assay Kit (POMG-25H) (BioAssay Systems) at 620 nm on a microplate reader, according to the manufacturer's protocol. Specific activity was calculated using a calibration curve and expressed as nmol Pi released/mg protein/min.
Each sample was run in triplicate. Remaining cells were lysed in 0.02% SDS in phosphate-buffered saline (PBS) and protein content determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific).

Ulivieri C., De Tommaso D., Finetti F., Ortensi B., Pelicci G., D'Elios M.M., Ballerini C, & Baldari C.T. (2019). A T Cell Suppressive Circuitry Mediated by CD39 and Regulated by ShcC/Rai Is Induced in Astrocytes by Encephalitogenic T Cells. Frontiers in Immunology, 10, 1041.

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Creation of Reporter Gene Constructs

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For creation of reporter gene constructs we combined a reporter with three different regulatory genomic fragments derived from the upstream region of AUTS2, containing MEF2C binding sites. We cloned genomic PCR products of the corresponding upstream regions (regulators) and of the HOXA9 gene, comprising exon1-intron1-exon2 (reporter), into the HindIII/BamHI and EcoRI sites, respectively, of the expression vector pcDNA3 downstream of the CMV enhancer [28 (link)]. The oligonucleotides used for the amplification of the AUTS2-regulators were obtained from Eurofins MWG. Their sequences were as follows: AUTS2-for1 5′-CCAAGCTTGTTGAAAGGTGACTGTAATTGC-3′, AUTS2-rev1 5′-AGGGATCCTGTGTTAACTTCTAGA GTGCTG-3′, AUTS2-for2 5′-CTAAGCTTTTTCCAAGT GAGCAAATAAGGAG-3′, AUTS2-rev2 5′-GGGGATCC TTAGGGCAAAGTTACAACAATG-3′, AUTS2-for3 5′-T GAAGCTTGTCAATAGTTCCTCTTGTGGTC-3′, AUT S2-rev3 5′-TTGGATCCTGGCACTTACTCTGTCCATT TC-3′. Introduced restriction sites used for cloning are underlined. Constructs were validated by sequence analysis (Eurofins MWG). Commercial HOXA9 and TBP assays were used for RQ-PCR to quantify the spliced reporter-transcript, corresponding to the regulator activity. A cotransfected commercial luciferase construct served as transfection control and was quantified by the Luciferase Assay System (Promega) using the luminometer Lumat LB9501 (Berthold Technologies, Bad Wildbad, Germany).

Nagel S., Pommerenke C., Meyer C., Kaufmann M., Drexler H.G, & MacLeod R.A. (2016). Deregulation of polycomb repressor complex 1 modifier AUTS2 in T-cell leukemia. Oncotarget, 7(29), 45398-45413.

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Quantifying Superoxide Release in Aortic Tissue

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The release of superoxide in intact aortic segments was measured by L-012 chemiluminescence, as previously described [24 (link)]. Aortas were carefully excised and placed in chilled, modified Krebs-HEPES buffer (pH 7.4; in mmol/L: NaCl 99.01, KCl 4.69, CaCl₂ 1.87, MgSO₄ 1.20, Na-HEPES 20.0, K₂HPO₄ 1.03, NaHCO₃ 25.0, and D(+)Glucose 11.1). Connective tissue was removed and aortas were cut into 2 mm segments. Chemiluminescence of aortic segments was assessed in scintillation vials containing Krebs-HEPES buffer with 100μmol/l L-012 over 10 minutes in a scintillation counter (Lumat LB 9501, Berthold) in 1 min intervals. The vessel segments were then dried and dry weight was determined. ROS release is calculated as relative chemiluminescence per mg aortic tissue and as percent of control.

Krogmann A.O., Lüsebrink E., Steinmetz M., Asdonk T., Lahrmann C., Lütjohann D., Nickenig G, & Zimmer S. (2016). Proinflammatory Stimulation of Toll-Like Receptor 9 with High Dose CpG ODN 1826 Impairs Endothelial Regeneration and Promotes Atherosclerosis in Mice. PLoS ONE, 11(1), e0146326.

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Validating miR-7-5p Regulation of OGT

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To verify that miR-7-5p can regulate OGT gene directly, we generated a Renilla luciferase reporter plasmid cloned downstream to a segment of the 3′UTR containing putative miR-7-5p binding sequences, which predicted the binding site, position 268–275 bp of OGT 3′UTR (NM_181672) using microRNA.org. The constructs were then transfected into NCI–H1299 cells with miR-7-5p mimic, mimic-negative control, and Renilla-Firefly reporter plasmid. After 48 h, the Renilla-Firefly luciferase activity was measured using a Lumat LB9501 instrument (Berthold, Bad Wildbad, Germany), and the results were normalized using the activity of the Firefly luciferase. All experiments were performed in triplicate.

Woo S.Y., Lee S.Y., Yu S.L., Park S.J., Kang D., Kim J.S., Jeong I.B., Kwon S.J., Hwang W.J., Park C.R, & Son J.W. (2020). MicroRNA-7-5p′s role in the O-GlcNAcylation and cancer metabolism. Non-coding RNA Research, 5(4), 201-207.

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Quantifying Superoxide Release in Aortic Tissue

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The release of superoxide in intact aortic segments was measured by L-012 chemiluminescence, as previously described (2 (link), 18 (link)). Aortas were carefully excised and placed in chilled, modified Krebs-HEPES buffer (pH 7.4; in mmol/L: NaCl 99.01, KCl 4.69, CaCl₂ 1.87, MgSO₄ 1.20, Na-HEPES 20.0, K₂HPO₄ 1.03, NaHCO₃ 25.0, and D-(+)-glucose 11.1). The connective tissue was removed and the aortas were cut into 2 mm segments. Chemiluminescence of the aortic segments was assessed in scintillation vials containing Krebs-HEPES buffer with 100 μmol/L L-012 over 10 min in a scintillation counter (Lumat LB 9501, Berthold, Bad Wildbad, Germany) in 1 min intervals. The vessel segments were then dried to determine their dry weight. ROS release was calculated as relative chemiluminescence per mg of aortic tissue as a percentage of the control.

Lüsebrink E., Goody P.R., Lahrmann C., Flender A., Niepmann S.T., Zietzer A., Schulz C., Massberg S., Jansen F., Nickenig G., Zimmer S, & Krogmann A.O. (2020). AIM2 Stimulation Impairs Reendothelialization and Promotes the Development of Atherosclerosis in Mice. Frontiers in Cardiovascular Medicine, 7, 582482.

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Bioluminescent Assay for Stress Response

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Three to four day old cells were diluted in assay medium at a concentration of 60 mg/ml and incubated in 6-well plates (5 ml/well) for 5 h shaking at 120 rpm in the dark at 26 °C. At different time points after elicitation, aliquots of 200 µl were added to 700 µl of 50 mM potassium phosphate buffer, pH 7.9, and light detection was performed in a cuvette luminometer (Lumat LB 9501, Berthold, Bad Wildbach, Germany) after automatic addition of 100 μl luminol (Sigma; 1.21 mM in phosphate buffer) and 100 μl 14 mM potassium hexacyanoferrate (Fluka), using an integration time of 10 s, at 430 nm.

Melcher R.L, & Moerschbacher B.M. (2016). An improved microtiter plate assay to monitor the oxidative burst in monocot and dicot plant cell suspension cultures. Plant Methods, 12, 5.

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Quantifying Transient Gene Expression in Tobacco Leaves

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At 3–4 d after transient co-transformation with GFP and LUC constructs, tobacco leaf fragments (1cm²) were cut off either for protein extraction or for confocal imaging. Total proteins were extracted from leaf fragments using the CCLR buffer of a Luciferase Assay System kit (Promega). GFP was quantified in protein extracts by fluorimetry (excitation at 485nm and emission at 535nm) using a Victor 3 plate spectrophotometer (Perkin Elmer). GFP fluorescence levels were normalized to LUC activity (Petit et al., 2001 (link)) determined using the Luciferase Assay System kit and a tube luminometer (Lumat LB 9501, Berthold).
GFP was imaged by confocal microscopy ensuring that the maximal fluorescence signal was not saturating the photomultiplier tubes, as described previously (Rausin et al., 2010 (link)). For GFP quantification, Z-optical sections of 45 and 65 µm were taken in randomly selected zones of leaf fragments. The resulting images were then stacked and three regions of interest were selected in each visible nucleus. GFP signal intensity was measured as the mean grey intensity in each region of interest, and the mean grey intensity for each construction was then computed.

Charlier J.B., Polese C., Nouet C., Carnol M., Bosman B., Krämer U., Motte P, & Hanikenne M. (2015). Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives. Journal of Experimental Botany, 66(13), 3865-3878.

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Quantifying ROS Production in HCASMC

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Production of reactive oxygen species by HCASMC was measured by L-012 chemiluminescence and 2',7'-dichlorofluorescin diacetate (DCF-DA) staining. L-012 stock solution (Wako Chemicals GmbH, Neuss, Germany) was diluted in PBS (1 : 100) and KH-HEPES-buffer (1 : 10), containing: NaCl 99.01 mM, KCl 4.69 mM, CaCl₂ 1.87 mM, MgSO₄ 1.20 mM, NaHEPES 20.0 mM, K₂HPO₄ 1.03 mM, NaHCO₃ 25.0 mM, D(+)glucose 11.1 mM, adjusted to pH 7.40 mM. HCASMC were stimulated with DMSO (0.1% (v/v)), E2 (10 nM), or E2 (10 nM) + GW9662 (1 µM). Cells were dissociated enzymatically with trypsin/EDTA (0.05% (w/v)), centrifuged (170 × g; room temperature; 5 min), and reconstituted in L-012 working solution. ROS catalysed a luminescent reaction that allowed for quantification in a scintillation counter (Lumat LB 9501, Berthold Technologies GmbH & Co. KG, Wildbad, Germany). Events were normalised to the cell count. Five-minute scintillation counts were used for statistical analyses.
In an additional approach, ROS were visualised by DCF-DA staining. HCASMC were stimulated with E2 (10 nM), E2 + GW9662 (1–30 µM), or DMSO (0.1% (v/v)) for 24 h. Hereafter, cells were exposed to 2 ml DCF-DA staining buffer, containing DCF-DA (10 µM) for 30 min at 37°C. Finally, cells were washed with staining buffer and microphotographic pictures were taken at an excitation wavelength of 485 nm and an absorption wavelength of 538 nm.

Jehle J., Tiyerili V., Adler S., Groll K., Nickenig G, & Becher U.M. (2020). Atheroprotective effects of 17β-oestradiol are mediated by peroxisome proliferator-activated receptor γ in human coronary artery smooth muscle cells. Archives of Medical Sciences. Atherosclerotic Diseases, 5, e118-e126.

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Dual-Luciferase Assay for Transfection

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Cells were seeded in 12-well plates and were transfected after 24 h with the indicated constructs. Luciferase activity was measured using the Lumat LB 9501 (Berthold Technologies, Bad Wildbad, Germany) luminometer and the Dual-Luciferase-Reporter Assay System (Promega) according to the manufacturer’s instructions. Firefly Luciferase values were normalized to Renilla luciferase activity and are shown as mean values ± SD.

Baumgart S., Chen N.M., Zhang J.S., Billadeau D.D., Gaisina I., Kozikowski A.P., Singh S., Fink D., Ströbel P., Klindt C., Zhang L., Bamlet W.R., Koenig A., Hessmann E., Gress T., Ellenrieder V, & Neesse A. (2016). GSK-3β governs inflammation-induced NFATc2 signaling hubs to promote pancreatic cancer progression. Molecular cancer therapeutics, 15(3), 491-502.

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Transcriptional Regulation Assay in HeLa Cells

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HeLa cells (American Type Culture Collection (ATCC) 2-CCL, Manassas, VA) were co-transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) with 0 - 2000 ng of effector plasmid, 200 - 600 ng of either the pG5CAT (Promega, Madison, WI), pG5Luc (Promega, Madison, WI), pGL3b/8XGli-lc-luc, or pBP100-GL2 reporter construct, and either 10 ng of Renilla control reporter (Promega, Madison, WI) or 400 ng of pSV40β-GAL control reporter (Promega, Madison, WI). Cell lysates were prepared 24 - 48 h after transfection. Luciferase activity was measured with a luminometer (Lumat LB9501, Berthold, Oak Ridge, TN) and was normalized using a Renilla control reporter (Promega, Madison, WI). CAT assays were performed by incubating lysates with ¹⁴C-chloramphenicol and n-Butyryl CoA. β-galactosidase activity was used to normalize the CAT activity in the lysate. The experiments were performed at least in triplicate and results expressed as a mean with standard deviation. Statistical significance was assessed using the Student’s t test.

Yoon J.W., Lamm M., Iannaccone S., Higashiyama N., Leong K.F., Iannaccone P, & Walterhouse D. (2015). P53 Modulates The Activity Of The GLI1 Oncogene Through Interactions With The Shared Coactivator TAF9. DNA repair, 34, 9-17.

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