Intracellular RSS levels of S. cerevisiae BY4742 cells were assessed by using a previously reported method [32 (link)]. In brief, BY4742 cells were suspended in 100 µL of reaction buffer (50 mM Tris-HCl and 1 mM sulfite (pH 7.5)) and incubated at 95 °C for 30 min. During incubation, sulfane sulfur atoms reacted with sulfite to form thiosulfate (S2O32−). Monobromobimane (mBBr) was then used to derivatize the formed thiolsulfate, and the derivative was quantified by using HPLC (LC-20A; Shimadzu, Kyoto, Japan) with a fluorescence detector (RF20A; Shimadzu, Kyoto, Japan). The obtained S2O32− derivative concentration represented the intracellular RSS level.
Rf 20a
Manufactured by Shimadzu
Sourced in Japan, United States
The RF-20A is a fluorescence detector developed by Shimadzu for high-performance liquid chromatography (HPLC) systems. It is designed to provide sensitive and reliable detection of fluorescent compounds. The RF-20A features adjustable excitation and emission wavelengths, allowing for the analysis of a wide range of fluorescent analytes.
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Lab products found in correlation
Lc 20ad, by Shimadzu (7 mentions)
Lc 20a, by Shimadzu (3 mentions)
Hplc system, by Shimadzu (3 mentions)
Lc 20at system, by Shimadzu (3 mentions)
Streptavidin, by AAT Bioquest (3 mentions)
Co 1560, by Jasco (3 mentions)
Pu 2080 plus, by Jasco (3 mentions)
Spd m20a, by Shimadzu (3 mentions)
Sil 20ac, by Shimadzu (2 mentions)
Lc 20at pump, by Shimadzu (2 mentions)
Lc 10at, by Shimadzu (2 mentions)
Spd 20a, by Shimadzu (2 mentions)
High performance liquid chromatography (hplc), by Shimadzu (2 mentions)
Sil 20a ht, by Shimadzu (2 mentions)
Cto 10as vp, by Shimadzu (2 mentions)
Cbm 20, by Shimadzu (2 mentions)
Lc 20at, by Shimadzu (2 mentions)
Dgu 20a5, by Shimadzu (2 mentions)
Cto 10a, by Shimadzu (1 mentions)
Labsolutions software, by Shimadzu (1 mentions)
61 protocols using rf 20a
1
Quantifying Intracellular RSS Levels in S. cerevisiae
2
HPLC-FL Analysis of Aflatoxin B1
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The analytical conditions for AFB1 were as described by Kushiro et al. [29 (link)]. The HPLC-FL system was composed of an HPLC column Capcell pack C18 UG120 (5 μm, 250 mm × 4.6 mm i.d.; Osaka Soda, Osaka, Japan), pump LC-20AD, column heater CTO-10A, autosampler SIL-20AC, fluorescence detector RF-20A, communication bus module CBM-20A, and LabSolutions software (Shimadzu, Kyoto, Japan). The mobile phase consisted of water, methanol, and acetonitrile (6:3:1), and the flow rate was 0.3 mL min−1. The column heater temperature was set at 40 °C. AFB1 was detected at wavelengths of 365 (extraction) and 450 nm (emission). The standard AF mix was diluted to generate the calibration curve. The limit of detection (LOD) and limit of quantitation (LOQ) for the analysis were approximately 0.25 and 0.74 ng mL−1, respectively, as calculated from the standard deviation of the y-intercept on the calibration curve.
3
Quantitative Tocopherol Extraction from Arabidopsis
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Leaf samples from each treatment and time point were quickly frozen with liquid nitrogen, and extractions were performed when all samples were collected. Details for extracting tocopherols from Arabidopsis leaf and seeds are listed below as short protocols following the methods section. HPLC separation and quantification were performed using a Waters YMC Carotenoid S-3 3.0 × 100 mm Column (cat # CT99S031003WT) and the following conditions. 12-min run, 0.8 ml/min gradient (Time, B %): 0–1 min, 100; 1–7.5 min, 76; 7.5–8 min, 0; 8–9.5 min, 0; 9.5–10 min, 100; 10–12 min, 100. 9.6-min run, 1.0 ml/min gradient (Time, B %): 0–0.8 min, 100; 0.8–6 min, 76.3; 6–6.4 min, 0; 6.4–7.6 min, 0; 7.6-8 min, 100; 8–9.6 min, 100. 8-min run, 1.2 ml/min gradient (Time, B %): 0–0.7 min, 100; 0.7–5.1 min, 76; 5.1–5.45 min, 0; 5.45–6.45 min, 0; 6.45–6.78 min, 100, 6.78–8 min, 100. Oven temperature was 30 °C and FLD (Fluorescence detector; Shimadzu model, RF-20A, cat# 228-45147-42) settings were Excitation Wavelength 290 nm and Emission Wavelength 325 nm with attenuation set to medium sensitivity. Solvent B: 90:10 v/v Methanol/Water; Solvent A: 100% MTBE (tert-Butyl methyl ether).
4
Quantification of Uremic Toxins by HPLC
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Total concentrations of UTs pCS, IS and IAA were quantified by HPLC method (High Performance Liquid Chromatography) with fluorescent detection. The ultrafiltered plasma was injected into HPLC system (Shimadzu Prominence, Tokyo, Japan) equipped with a manual injector model 7125 Rheodyne, a quaternary pump (Shimadzu LC-20AD, Tokyo, Japan), controlled by LC Solution software (Shimadzu, Tokyo, Japan) and equipped with a fluorescence detector Shimadzu RF-20A. Identification of UTs followed the protocols of the published methods [17 (link),18 (link)]. The toxins were separated by a C8 column (Phenomenex, Luna 5 μm, 100A, 150 × 4.6 mm) using concentration gradient eluted with 50 mM ammonium formate pH 3.0 and methanol whose proportion increased from 35% to 70% along the run, at a flow rate of 0.7 mL/min. During the run, the fluorescence wavelengths varied as follows: λexc = 280 nm and λem = 383 nm to IS and IAA, λexc = 265 nm and λem = 290 nm to pCS.
5
Quantification of Urinary 1-Aminopyrene
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Urinary 1-AP levels were measured from 2 h urine samples. One milliliter of 10 M hydrochloric acid was added to each 10 mL urine sample, and the urine was stirred in a 90-°C water bath for 2 h. After adjusting the pH to 7.0–8.0, the supernatant was extracted with Sep-Pak cartridge (C18, 3 mL, 200 mg, Waters, Milford, UK). Before extraction, it was pre-conditioned with 5 mL of methanol and 5 mL of water. After loading the sample, it was washed with 5 mL of 20% methanol in water and then extracted with 4 mL of 100% methanol. The extract was dried with N2 gas and dissolved in 200 μL of methanol. The extract was analyzed by injecting 20 μL into the HPLC system equipped with a fluorescence detector (Shimadzu, RF-20A, Kyoto, Japan). A reverse phase-amide column (Ascentis RP-Amide, 25 cm × 4.6 mm, 5 μm, SUPELCO, Bellefonte, PA, USA) was used. The mobile phase was composed of methanol and 50 mM sodium acetate buffer pH 7.2 (80:20, v/v) at a flow rate of 1.0 mL/min, and the excitation and emission wavelengths were 254 nm and 425 nm, respectively (Fig. 1 ). Urinary 1-AP concentration was corrected using the urinary creatinine level.![]()
Chromatograms for 1-aminopyrene (
6
Quantification of Tocopherols in Lecithins by HPLC
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Tocopherols were quantitated by normal phase HPLC [39 (link)] using a mixture of n-hexane and 1,4-dioxane (95:5, v/v) as the mobile phase at a flow rate of 1 mL/min. The lecithins (0.01 g) were dissolved in 2 mL of n-hexane and aliquoted into HPLC vials using a 0.2 µm syringe filter. Each sample (20 μL) was injected into Shimadzu Prominence-i LC-2030C HPLC system equipped with a Supelcosil LC Diol column (L × i.d. = 25 cm × 4.6 mm, 5 µm particle size). The effluent was monitored with a fluorescence detector (Shimadzu RF—20A) with an excitation wavelength at 290 nm and emission wavelength at 330 nm. The tocopherols were identified using retention times and were quantified with standard curves (peak areas) obtained from authentic standards.
7
Quantifying Anserine and Carnosine in Kidney Tissue
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Anserine and carnosine concentrations in kidney tissue of WT, Cndp1-KO and Hsp70-KO mice were measured fluorometrically using HPLC as described previously [32 (link)]. Next, 50 mg of frozen renal tissue was homogenized, diluted and subsequently derivatized with carbazole-9-carbonyl chloride (CFC) and measured by fluorescence detection (RF-20A, Shimadzu, Kyoto, Japan; Jupiter column C18, 300 Å, 5 µm particle size, 250 × 4.6 mm, Phenomenex, Aschaffenburg, Germany). Mobile phase consisted of a binary gradient with 82% solution A (50 mmol/L acetate buffer in distilled water; pH 4.37) and 18% solution B (acetonitrile, methanol and tetrahydrofuran 70:25:5 (v/v/v)).
8
Quantification of Amino Acid Neurotransmitters
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Amino acid transmitters were extracted using brain tissue extract liquid (water: acetonitrile=2: 3); we added 100 μl/mg into MNTB tissues, fully ground the tissues, and ultrasonically extracted the amino acid transmitters in an ice bath for 20 min. The supernatant was obtained by 15 000 rpm centrifugation at 4°C for 10 min, followed by centrifugation at 4°C 15 000 r/min for 5 min; the supernatant containing all amino acid transmitters was then diluted to 1: 50 for use. HPLC-FLD was conducted in a High-Performance Liquid Chromatography device (Sykam, Germany) and a Fluorescence Detector device (Shimadzu RF-20A, Japan), using an amino acid analysis column (Eclipse AAA 4.6×150 mm, 5 μm) and Phthalic dicarboxaldehyde (OPA) pre-column derivatization. The mobile phase was: A: buffer solution: methanol: tetrahydrofuran (V: V: V)=400: 95: 5; and B: buffer solution: methanol (V: V)=120: 380, gradient elution. The buffer solution was 20 mM of sodium acetate solution (pH=7.2). The flow rate was 0.8 ml/min, using the fluorescence detector to detect the amino acids-derived compound after separation, with λex=340 nm and λem=455 nm.
9
Quantification of Salicylic and Jasmonic Acids
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The quantification of SA and SA glucoside (SAG) was performed as described previously with a minor modification (Seo et al. 1995 (link)). Briefly, three leaf disks were frozen and ground using liquid nitrogen. SA and SAG were extracted with 90% methanol, and SAG was converted to SA by β-glucosidase treatment. After separation by HPLC (Shimadzu) with an ODS column (μ-Bondasphere C18, 150 mm�ID3.9 mm, 5 μm, 100A; Waters), SA levels were determined using a fluorescence detector (RF-20A; Shimadzu) with an excitation wavelength of 313 nm and an emission wavelength of 405 nm.
JA quantification was performed as described previously (Kojima et al. 2009 (link), Shinozaki et al. 2015 (link)). Briefly, leaf samples (one leaf per sample) were frozen and ground using liquid nitrogen, and freeze dried. JA was extracted and determined using an ultra-HPLC-Q-Exactive™ system (Thermo Scientific) using an ODS column (AQUITY UPLC BEH C18, 1.7 μm, 2.1�100 mm; Waters) as described (Shinozaki et al. 2015 (link)).
JA quantification was performed as described previously (Kojima et al. 2009 (link), Shinozaki et al. 2015 (link)). Briefly, leaf samples (one leaf per sample) were frozen and ground using liquid nitrogen, and freeze dried. JA was extracted and determined using an ultra-HPLC-Q-Exactive™ system (Thermo Scientific) using an ODS column (AQUITY UPLC BEH C18, 1.7 μm, 2.1�100 mm; Waters) as described (Shinozaki et al. 2015 (link)).
10
HPLC Quantification of Serum PCS and IS
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PCS and IS in patients’ serum were quantified by HPLC as previously described [56 (link)]. The supernatant was ultrafiltered with a 30 kDa cutoff membrane Amicon Ultra (Millipore, Burlington, MA, USA). Chromatographic analysis was done using a Shimadzu Prominence system equipped with a Rheodyne injector, a quaternary pump (Shimadzu LC-20AD), controlled by the LC Solution software and a fluorescence detector (Shimadzu RF-20A). The toxins were separated on a C8 Luna column (5 μm, 100 Å, 150 × 4.6 mm) (Phenomenex, Torrance, CA, USA), eluted with 50 mM ammonium formate, pH = 3.0 and a methanol gradient of 35–70% (v/v), at a flow rate of 0.7 mL/min. The toxins were detected using fluorescence (PCS: λexc = 265 nm and λem = 290 nm [57 (link)] and IS: λexc = 283 nm and λem = 380 nm) [58 (link),59 (link)].
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