Fluoromax 4c

Manufactured by Horiba

The Fluoromax-4C is a fluorescence spectrophotometer designed for high-performance fluorescence measurements. It features a xenon arc lamp as the excitation source and a photomultiplier tube detector. The Fluoromax-4C is capable of performing steady-state fluorescence, phosphorescence, and luminescence measurements.

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Lab products found in correlation

Model f 3004, by Horiba (2 mentions) Methionine, by Merck Group (2 mentions) Adenosine, by Merck Group (2 mentions) S adenosylmethionine, by Merck Group (2 mentions) S adenosylhomocysteine, by Merck Group (2 mentions) Spectramax m series, by Molecular Devices (1 mentions) Uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) A7007, by Merck Group (1 mentions) Picoharp 300, by PicoQuant (1 mentions) Infinity3 3ur, by Teledyne (1 mentions) Flash 4.0 lt, by Hamamatsu Photonics (1 mentions)

Close spelling variants of this product

Fluoromax 4C spectrofluorometer (3 citations) Yvon Fluoromax 4C-L (1 citations)

10 protocols using fluoromax 4c

Fluorescent RNA Visualization Assay

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Purified RNAs were diluted to 1 μM in buffer containing 100 mM KCl, 1 mM MgCl₂, 40 mM HEPES (pH 7.5). RNAs were then heated up to 75°C for 5 min and then cooled to room temperature. RNA solutions were then mixed with an equal volume of the same buffer containing 20 μM DFHBI-1T or DFHO (synthesized (Song et al., 2014 (link), 2017 (link)) or Lucerna 410-1mg). Samples contained a final concentration 0.5 μM RNA and 10 μM DFHBI-1T or DFHO, unless otherwise noted. After 1 h incubation, fluorescence signal of each sample was measured using a Fluoromax-4C (Horiba Scientific) with 460 nm excitation and 505 nm emission, 5 nm slit widths, and 0.1 s integration time. Background signal was detected with the RNA-free sample containing the same concentration of DFHBI-1T in 100 mM KCl, 1 mM MgCl₂, 40 mM K-HEPES (pH 7.5). The detected background signal was subtracted from the signal obtained from each RNA-containing sample. Some fluorescence data was collected using a SpectraMax M-series plate reader (Molecular Devices) (figures 5 B and C, S1 B, C, F, and G, S3, S4, and S5 A and B). Results are shown for n=3 biological replicates, unless otherwise stated. No blinding was used in these experiments.

Moon J.D., Wu J., Dey S.K., Litke J.L., Li X., Kim H, & Jaffrey S.R. (2021). Naturally occurring three-way junctions can be repurposed as genetically encoded RNA-based sensors. Cell chemical biology, 28(11), 1569-1580.e4.

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Structural Characterization of QDs Assemblies

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Structural
characterization of the QDs assemblies were carried out using a TEM
at 120 kV. Absorbance spectra were measured using a UV–vis
spectrophotometer (Thermo-Fisher) and the PL spectra were measured
using a FluoroMax-4C (Horiba Jobin Yvon). The extinction coefficient
of QD was estimated using the first extinction absorption peak and
calculated according to the empirical formula from refs (59 and 60 (link)). The quantum yield of QD was
determined using the relative quantum yield determination method with
rhodamine 101 in spectroscopic-grade ethanol as standard in refs (59 and 61 (link)). The Förster distances
and FRET efficiencies were calculated according to ref (62 (link)). (details are available
in SI).

Wei X., Chen C., Zhao Y., Harazinska E., Bathe M, & Hernandez R. (2022). Molecular Structure of Single-Stranded DNA on the ZnS Surface of Quantum Dots. ACS Nano, 16(4), 6666-6675.

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Aptamer Melting Temperature Profiling

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The fluorescence of the aptamers was measured at different temperatures using a Fluoromax 4C (Horiba) with a Peltier-controlled temperature attachment (Model F-3004, Horiba). The fluorescence of the aptamer–dye complex was recorded in 3 °C increments from 1 to 70 °C, with a 10 min incubation at each interval. Fluorescence intensity spectra (excitation at 617 nm, emission at 650–750 nm) were recorded and the baseline was subtracted from raw fluorescence values. Melting curves were fitted in Origin Pro 9 software using the Boltzmann sigmoidal equation F = F_min + (F_max − F_min)/(1 + exp((T_t − T)/s)), where F refers to fluorescence (λ = 660 nm) and F_min and F_max are the minimum and maximum fluorescence, respectively, T is temperature, T_t is the transition temperature, and s is a fitting parameter. T_t values presented here are average of three independent experiments. Fatty acid vesicles are known to be thermostable across this temperature regime^{79 (link)}. For samples containing vesicles, the background fluorescence (λ = 660 nm) of a control sample containing dye in the presence of vesicles (without aptamer) was subtracted from the fluorescence of vesicles with aptamer-bound dye at each temperature.

Saha R., Verbanic S, & Chen I.A. (2018). Lipid vesicles chaperone an encapsulated RNA aptamer. Nature Communications, 9, 2313.

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Biosensor RNA Fluorescence Assay

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Biosensor RNA was transcribed in vitro using the AmpliScribe™ T7-Flash™ transcription kit (Lucigen ASF3507) as describe above. RNAs were column purified and diluted to 1 μM in a buffer prepared at room temperature containing 10 μM DFHBI-1T, 100 mM KCl, 0.2 mM MgCl₂, 40 mM HEPES, pH 7.4. S-adenosyl methionine, S-adenosyl homocysteine, adenosine, or methionine (all from Sigma Aldrich) were added to samples at 100 μM and fluorescence signal was measured at RT using a Fluoromax-4C (Horiba Scientific) with 470 nm excitation and 505 nm emission, 5 nm slit widths, and 0.1 s integration time.
To measure biosensor activation rate, the biosensor RNA was prepared to 1 μM in a buffer containing 10 μM DFHBI-1T, 100 mM KCl, 5 mM MgCl₂, 40 mM HEPES, pH 7.4 in a constantly stirring cuvette at 37 °C. Fluorescence was collected by kinetics acquisition with 0.2 s integration time and after 1 min of collecting background signal, 100 μM of S-adenosyl methionine was quickly added to the cuvette.

Litke J.L, & Jaffrey S.R. (2019). Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nature biotechnology, 37(6), 667-675.

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Sensing S-Adenosyl Methionine with Broccoli Aptamer

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Purified RNAs dissolved in nuclease-free water were heated up to 75°C for 5 min and cooled down to 4°C for 5 min before incubating with the in dicated buffer. RNAs were diluted to a final concentration of 1 μM in buffer solution containing 100 mM KCl, 1 mM MgCl₂, 40 mM K-HEPES (pH 7.5) and 10 μM DFHBI-1T (synthesized (Song et al., 2014 (link)) or Lucerna 410-1mg). To test if the circularly permuted SAM aptamer-Broccoli fusion RNA enables to sense SAM, a final concentration of 0.1 mM SAM (Sigma-Aldrich A7007) was added to the prepared RNA sample. After 1 h incubation at 37°0, fluorescence signal of the sample was measured at 37°C using a Fluoromax-4C (Horiba Scientific) with 470 nm excitation and 505 nm emission, 5 nm slit widths, and 0.1 s integration time. Background signal was subtracted from the signal obtained from each RNA sample measurement. To obtain background signal, an RNA-free sample containing 10 μM DFHBI-1T in 100 mM KCl, 1 mM MgCl₂, 40 mM K-HEPES (pH 7.5) was used, and was subjected to fluorescence measurement at 37°C.

Kim H, & Jaffrey S.R. (2019). A fluorogenic RNA-based sensor activated by metabolite-induced RNA dimerization. Cell chemical biology, 26(12), 1725-1731.e6.

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Biosensor RNA Fluorescence Assay

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Litke J.L, & Jaffrey S.R. (2019). Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nature biotechnology, 37(6), 667-675.

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Synthetic Melanin Spectral and Lifetime Analysis

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Dark synthetic melanin solution (1 g/ml; Sigma-Aldrich) was prepared to produce a range of melanin concentrations (0.01 mg/ml, 0.025 mg/ml, 0.05 mg/ml, 0.1 mg/ml) in MilliQ water. The preparation of the solubilized synthetic melanin was based on a protocol used by Riesz et al.^{73 (link)}. The pH of the solutions was adjusted using 1 M NaOH to around 11.5 with constant stirring to aid solubility.
Single point measurements of spectral emission and fluorescence lifetime were acquired using the Fluoromax-4C spectrofluorometer Horiba Scientific. For spectral emission single point measurements, synthetic melanin samples (0.01 mg/ml, 0.025 mg/ml, 0.05 mg/ml, 0.1 mg/ml) were excited at 390 nm and spectral emission were collected between 405–700 nm. The fluorescence lifetime single point measurements were acquired at 370 nm excitation, the only laser source for this module.

Sitiwin E., Madigan M.C., Gratton E., Cherepanoff S., Conway R.M., Whan R, & Macmillan A. (2019). Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques. Scientific Reports, 9, 18585.

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FRET-based RNA Melting Curve Analysis

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A stock solution (100 μM) of the FRET-labeled stem-loop RNA 11 was heated to 75 °C for 5 min and slowly cooled down to room temperature to anneal the RNA structure. The fluorescence of the dye-labelled RNA was measured at different temperatures using a Fluoromax 4C (Horiba) with a Peltier-controlled temperature attachment (Model F-3004, Horiba). The fluorescence of the labeled RNA was recorded in 3 °C increments from 10 to 67 °C, with a 10 min incubation at each interval. Fluorescence intensity spectra (excitation at 450 nm, and emission spectrum between 480 and 700 nm) were recorded. Melting curves were fitted in Origin Pro 9 software using the Boltzmann sigmoidal equation R = R_min + (R_max − R_min)/(1 + exp((T_t − T)/s)), where R refers to normalized fluorescence intensity ratio of F_520nm/ F_590nm, and R_min and R_max are the minimum and maximum values, respectively, T is temperature, T_t is the transition temperature, and s is a fitting parameter. T_t values presented here are an average of three independent experiments.

Peng H., Lelievre A., Landenfeld K., Müller S, & Chen I.A. (2021). Vesicle encapsulation stabilizes intermolecular association and structure formation of functional RNA and DNA. Current biology : CB, 32(1), 86-96.e6.

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Corn-SAM Sensor RNA Fluorescence Assay

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The Corn-SAM sensor RNAs were diluted to 1 μM in buffer solution containing 100 mM KCl, 0.5 mM MgCl₂, 40 mM K-HEPES (pH 7.5) and 10 μM DFHO. To test if fluorescence signal of the Corn-SAM sensor is specifically activated by SAM, 0.1 mM or 0.5 mM of the following analyte, SAM (Sigma-Aldrich A7007), SAH (Sigma-Aldrich A9384), adenosine (Sigma-Aldrich 9251) or methionine (Sigma-Aldrich M9625), was added to the prepared RNA samples. Then, the samples were incubated at 37°C for 1 h. The fluores cence signal of each sample was measured at 37°C using a Fluoromax-4C (Horiba Scientific) wi th 505 nm excitation and 545 nm emission, 5 nm slit widths, and 0.1 s integration time. Background signal was subtracted from the signal obtained from each RNA sample measurement. To obtain background signal, an RNA-free sample containing 10 μM DFHO in 100 mM KCl, 0.5 mM MgCl₂, 40 mM K-HEPES (pH 7.5) was used, and was subjected to fluorescence measurement at 37°C.

Kim H, & Jaffrey S.R. (2019). A fluorogenic RNA-based sensor activated by metabolite-induced RNA dimerization. Cell chemical biology, 26(12), 1725-1731.e6.

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Fluorescence-based Cortisol Nanosensor Characterization

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The fluorescence spectra of aptamer–QD@MNP and antibody–QD@MNP nanosensors were measured in a solution to characterize cortisol detection using a spectrofluorometer (FluoroMax-4c, Horiba) with 380 nm wavelength excitation. Steroid hormone-induced QD fluorescence quenching was performed on a glass substrate and analyzed using an inverted fluorescence microscope (Olympus IX73) through a 40× objective lens under UV excitation with a spectral range from 350 to 400 nm produced by a light-emitting diode (LED) excitation (X-Cite 120) and a filter set (DAPI-50LP). The fluorescence signals were characterized by a monochromic sCMOS camera (Hamamatsu Flash 4.0 LT) on the microscope. The color fluorescent images were captured by a color charge-coupled device (CCD) camera (Lumenera Infinity3–3UR). The fluorescent images were taken with background subtraction, and the same setting was used throughout the experiment. Fluorescence lifetime measurements were accomplished using a time-correlated single-photon counting (TCSPC) system (Picoharp 300, PicoQuant) and 375 nm UV pulsed laser excitation.

Liu Y., Wu B., Tanyi E.K., Yeasmin S, & Cheng L.J. (2020). Label-Free Sensitive Detection of Steroid Hormone Cortisol Based on Target-Induced Fluorescence Quenching of Quantum Dots. Langmuir : the ACS journal of surfaces and colloids, 36(27), 7781-7788.

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