Rf 2000 fluorescence detector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The RF 2000 Fluorescence Detector is a laboratory instrument designed to detect and measure fluorescence signals. It is a core component in various analytical techniques that rely on fluorescence-based detection. The RF 2000 Fluorescence Detector provides high sensitivity and selectivity in the measurement of fluorescent compounds.

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Lab products found in correlation

Rs variable wavelength ultimate 3000 detector, by Thermo Fisher Scientific (2 mentions) Chromeleon software, by Thermo Fisher Scientific (1 mentions) Ultimate 3000 autosampler, by Thermo Fisher Scientific (1 mentions) Tryptophan trp, by Merck Group (1 mentions) Kynurenic acid kyna, by Merck Group (1 mentions) Hc c18 column, by Agilent Technologies (1 mentions) Luna c18 100a column, by Phenomenex (1 mentions) Ultimate 3000 pump, by Thermo Fisher Scientific (1 mentions) Hp 5890 series 2 gc, by Hewlett-Packard (1 mentions) Rxi 5sil ms, by Restek (1 mentions) Agilent hc c18, by Agilent Technologies (1 mentions) Ultimate 3000 analytical systems, by Thermo Fisher Scientific (1 mentions)

5 protocols using rf 2000 fluorescence detector

HPLC Analysis of TRP, KYN, and KYNA

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TRP, KYN, and KYNA were analyzed by high-performance liquid chromatography (HPLC), according to the protocol of Zhao et al. (2010), with minor adjustments [38 (link)]. Briefly, to each milk and serum sample, 6% HClO₄ was added and the sample was centrifuged at 12,000× g for 30 min at 4 °C. The resulting supernatant was used for HPLC with the Dionex P680 Pump, UltiMate 3000 Autosampler with column compartment (Sunnyvale, CA, USA). Agilent HC-C18(2) column (250 × 4.6 mm i.d.; 5-µm particle size) was used coupled to RS Variable Wavelength UltiMate 3000 Detector (Dionex, Sunnyvale, CA, USA) set at wavelengths 250 nm for TRP, 365 nm for KYN, and RF 2000 Fluorescence Detector (Dionex) set at excitation wavelength of 348 nm and emission at 398 nm for KYNA determination. The mobile phase was a mixture of 20-mM NaAc, 5-mM ZnAc₂, and 4% acetonitrile for which the flow rate was 1.0 mL/min. Control of the HPLC system and data collection were performed with the Chromeleon software (Dionex).
A full description and validation of the method is provided in the paper, by Zhao et al. (2010) [38 (link)].
IDO activity was calculated as a ratio by dividing the content of KYN (in µM multiplied by 1000) by that of TRP (in µM) [39 (link)].

Bochniarz M., Piech T., Kocki T., Iskra M., Krukowski H, & Jagielski T. (2021). Tryptophan, Kynurenine and Kynurenic Acid Concentrations in Milk and Serum of Dairy Cows with Prototheca Mastitis. Animals : an Open Access Journal from MDPI, 11(12), 3608.

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HPLC Analysis of Tryptophan Metabolites

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Tryptophan (TRP), L-kynurenine sulfate (KYN), and kynurenic acid (KYNA) were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA) and used as a standard. HPLC-grade chemicals were purchased from J.T. Baker Chemicals (Aventor Performance Materials Poland S.A., Gliwice, Poland) or Sigma-Aldrich. TRP, KYN, and KYNA were analyzed by high-performance liquid chromatography (HPLC), according to the protocol of Zhao et al. (2010) [56 (link)], with minor modifications. Briefly, each serum sample was added 6% HClO₄ and centrifuged at 12,000× g for 30 min at 4 °C. The resulting supernatant was applied to an HPLC system consisting of the Dionex P680 Pump, UltiMate 3000 Autosampler with column compartment, RS Variable Wavelength UltiMate 3000 Detector, and RF 2000 Fluorescence Detector (Dionex, Sunnyvale, CA, USA). An Agilent HC-C18 column (250 × 4.6 mm i.d.; 5-µm particle size) was used. The mobile phase was composed of 20 mM sodium acetate, 5 mM zinc acetate, and 4% acetonitrile; the flow rate was 1.0 mL/min. Detectors were set at wavelengths 250 nm for TRP, 365 nm for KYN, and at excitation wavelength of 348 nm and emission at 398 nm for KYNA determination. Control of the HPLC system and data analysis were performed with the Chromeleon software (Dionex^TM Chormeleon^TM version 7.2.6 (10049), serial number 117836).

Turska-Kozłowska M., Pedraz-Petrozzi B., Paluszkiewicz P, & Parada-Turska J. (2024). Different Kynurenine Pathway Dysregulation in Systemic Sclerosis in Men and Women. International Journal of Molecular Sciences, 25(7), 3842.

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Quantitative Determination of KYNA

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The quantitative determination of KYNA was performed as described by Turski et al. [31 (link),35 (link)]. The samples of dried, pulverized plant material or flax seeds were mixed with distilled water (1:10, w/v) and homogenized. In the case of KYNA isolation from flax seeds, they were ground in a laboratory mill (ZBPP P008M Sp. z o.o. Bydgoszcz, Poland) before the procedure. After centrifugation (5000 rpm, 10 min), the obtained supernatant was acidified with trichloroacetic acid (50%) and centrifuged (12,000 rpm, 10 min). Then samples were mixed with 0.1 N HCl and the fraction containing KYNA was separated using ion exchange chromatography with Dowex 50 columns and prewashed with 0.1 N HCl. Then, the column was washed using 1 mL of 0.1 N HCl and 1 mL of distilled water, and KYNA elution was performed with distilled water (3 mL). KYNA level was analysed using the HPLC method (Dionex HPLC system; C18 reverse-phase column) and quantified fluorometrically (Dionex RF2000 fluorescence detector; excitation 350 nm; emission 404 nm). The mobile phase consisted of 50 mM sodium acetate and 250 mM zinc acetate (pH 6.2) containing 5% acetonitrile. The flow rate was 1.0 mL/min. KYNA purchased from Sigma was used as a reference for HPLC.

Wróbel-Kwiatkowska M., Turski W., Silska G., Rakicka-Pustułka M., Dymińska L, & Rymowicz W. (2024). Determination of Bioactive Compound Kynurenic Acid in Linum usitatissimum L.. Molecules, 29(8), 1702.

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GC-MS and LC-Fluorescence Analysis of MXC and BaP

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MXC in the organic phase of samples from the in vitro incubations and in DCM extracts of water samples from the in vivo bioconcentration tests were analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS system consisted of a HP5890 series II GC coupled to a HP5972 mass sensitive detector (Hewlett-Packard). Separations were performed on 5% phenyl polysilphenylene-siloxane capillary columns (BPX-5, 30 m length x 0.25 mm i. d, 0.25 µm d_f, SGE). Analysis of BaP in ACN extracts of samples from the in vitro incubations and in ACN/water extracts of water samples from the in vivo bioconcentration tests was carried out by liquid chromatography (LC)-fluorescence detection on a HPLC Summit system equipped with an Ultimate 3,000 pump and a RF 2000 fluorescence detector (Dionex). Separations were performed on a Luna C18 100A column (Phenomenex, 250 mm × 4.6 mm, 5 μm particle size). Toluol extracts of fish samples from the in vivo bioconcentration studies were analyzed for MXC and BaP by GC-MS using a Varian Bruker 450-GC coupled to Varian Bruker 320-MS. Separations were performed on 1,4-bis(dimethylsiloxy)phenylene dimethyl polysiloxane capillary columns (Rxi®-5Sil MS, 20 m length x 0.18 mm i. d, 0.18 µm d_f, Restek). A detailed description of the instrument conditions used for GC-MS and LC-fluorescence analysis is provided in appendices (Supplementary Appendix A.1).

Bischof I., Arnot J.A., Jürling H., Knipschild G., Schlechtriem C., Schauerte A, & Segner H. (2022). In vitro biotransformation assays using fish liver cells: Comparing rainbow trout and carp hepatocytes. Frontiers in Toxicology, 4, 1021880.

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Tryptophan, Kynurenine, and KYNA Quantification

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Tryptophan, KYN, and KYNA concentrations were measured according to the method of Zhao et al. [47 (link)]. In brief, the studied substances were analyzed by the HPLC system (The UltiMate 3000 Analytical systems (Thermo Fisher Scientific, Waltham, MA, USA)). The samples were separated on an analytical column (Agilent HC-C18; 250 × 4.6 mm, inner diameter). The mobile phase was composed of 20 mmol/L NaAc, 3 mmol/L ZnAc₂, and 7% acetonitrile. It was pumped at a flow rate of 1 mL/min, and the volume per injection was 100 μL. The wavelength of the UV detector was set at 250 nm for tryptophan, 365 nm for KYN, and the RF 2000 Fluorescence Detector (Dionex) was set at an excitation wavelength of 348 nm and emission at 398 nm for KYNA determination. Chromeleon 7.2 software was used to control the HPLC system and record chromatographic data.

Marszalek-Grabska M., Gawel K., Kosheva N., Kocki T, & Turski W.A. (2023). Developmental Exposure to Kynurenine Affects Zebrafish and Rat Behavior. Cells, 12(18), 2224.

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