The whole cell lysate was collected following treatment with RIPA buffer (Millipore, billerica, MA, USA) supplemented with the protease inhibitor PMSF. The total protein concentration of the samples was measured using a commercially available BCA protein assay kit (Spectra Max190; Molecular Devices, San Jose, CA, USA). Enzyme assays for SIRT1 activity were performed as per the manufacturer's instructions (Sigma‐Aldrich). The plates were read with a fluorescent spectrophotometer (Spectra Max190; Molecular Devices) at an excitation wavelength of 340 nm and emission wavelength of 430 nm.
Spectramax 190
Manufactured by Molecular Devices
Sourced in United States, Japan, China, United Kingdom, Germany, Sweden
The SpectraMax 190 is a microplate reader designed for absorbance-based measurements. It features a xenon flash lamp as the light source and can perform absorbance readings across a wavelength range of 190 to 850 nanometers. The instrument can accommodate 6- to 384-well microplates.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (21 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (18 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (14 mentions)
Softmax pro software, by Molecular Devices (13 mentions)
Cck 8, by Dojindo Laboratories (11 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (9 mentions)
Elisa kit, by R&D Systems (8 mentions)
Cck 8 solution, by Dojindo Laboratories (7 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (7 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (7 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (5 mentions)
Cck 8, by Molecular Devices (5 mentions)
Transwell, by Corning (5 mentions)
Cck 8 assay, by Dojindo Laboratories (5 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (5 mentions)
Softmax pro 7, by Molecular Devices (5 mentions)
96 well plate, by Thermo Fisher Scientific (4 mentions)
Stop solution, by Solarbio (4 mentions)
Tmb substrate solution, by Solarbio (4 mentions)
1 147 protocols using spectramax 190
1
SIRT1 Activity Quantification via Fluorescence
2
Quantifying Alzheimer's Biomarkers in CSF
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Total Aβ protein concentration in CSF was determined using a bicinchoninic acid assay kit (Zhang et al. 2019 (link)) (P0010S, Beyotime Biotechnology, Nantong, Jiangsu Province, China). The optical densities were recorded using an enzyme mark instrument (SPECTRAMAX 190, Molecular Devices Corp., Sunnyvale, CA, USA) at 450 nm wavelength. A calibration curve was thus generated to calculate the protein concentrations in CSF.
The HA level in CSF was determined using an enzyme-linked immunosorbent assay kit for human HA according to the manufacturer’s instruction (Yoo et al. 2018 (link)) (ml064280, Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). The sample treatment was the same as with total Aβ protein. The optical density developed in proportion to the amount of specific cytokine was recorded using an enzyme mark instrument (SPECTRAMAX 190, Molecular Devices Corporation, Sunnyvale, CA, USA) at 450 nm wavelength. A calibration curve was then generated to calculate the concentration of HA in CSF.
The HA level in CSF was determined using an enzyme-linked immunosorbent assay kit for human HA according to the manufacturer’s instruction (Yoo et al. 2018 (link)) (ml064280, Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). The sample treatment was the same as with total Aβ protein. The optical density developed in proportion to the amount of specific cytokine was recorded using an enzyme mark instrument (SPECTRAMAX 190, Molecular Devices Corporation, Sunnyvale, CA, USA) at 450 nm wavelength. A calibration curve was then generated to calculate the concentration of HA in CSF.
3
Serum IgG, IgM and Antioxidant Biomarkers
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Concentrations of serum IgG and IgM were determined with porcine IgG and IgM ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with a microplate reader (SpectraMax®190, Molecular Devices, USA) at 450 nm. The logistic curves of IgG and IgM were built according to the manufacturer's instructions, and used the analysis software of ELISA calc. The limits for IgG and IgM concentrations were 0.3–90 mg/mL and 0.1–30 mg/mL, respectively. Coefficients of intra- and inter-sample variations were all <10 and 12% for IgG and IgM, respectively.
The content of reduced glutathione (GSH) and malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were assayed using specific assay kits (Product code: A006-2, A003-1, A001-3, Nanjing Institute of Jiancheng Biological Engineering, Nanjing, China) according to the manufacturer's instruction. A microplate reader (SpectraMax®190, Molecular Devices, USA) with the absorbance of 405, 532, and 450 nm for GSH, MDA, and SOD, respectively was recommended to read the numbers. Parallel determination was conducted for each sample.
The content of reduced glutathione (GSH) and malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were assayed using specific assay kits (Product code: A006-2, A003-1, A001-3, Nanjing Institute of Jiancheng Biological Engineering, Nanjing, China) according to the manufacturer's instruction. A microplate reader (SpectraMax®190, Molecular Devices, USA) with the absorbance of 405, 532, and 450 nm for GSH, MDA, and SOD, respectively was recommended to read the numbers. Parallel determination was conducted for each sample.
4
Quantifying Adipocyte-Derived Free Fatty Acids and Triglycerides
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To detect the free fatty acid levels in each media, CM was obtained from adipocytes cultured alone or indirectly co-cultured with A549 cells at 37°C and subsequently analyzed using a non-esterified fatty acid quantification kit (cat. no. A042-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) under a microplate reader (SpectraMax 190; Molecular Devices, LLC) at 440 nm, according to the manufacturer's protocol. To detect intracellular TGs, following indirect co-culture, adipocytes or A549 cells were centrifuged at 550 × g for 10 min at RT, and sonicated at 300 W, 5 sec/beat, 30 sec of interval, 3–5 repeats in an ice bath. The intracellular TG content was determined using a glycerol-3-phosphate oxidase/phenol + aminophenazone enzymatic kit (cat. no. A110-1; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol. The TG and FFA content were determined at 510 and 440 nm, respectively, under a microplate reader (SpectraMax 190; Molecular Devices, LLC).
5
SIRT1 Activity Assay in Cell Lysates
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Whole cell lysate was collected following treatment with RIPA buffer (Millipore, MA) supplemented with the protease inhibitor PMSF. Total protein concentration of the samples was measured using a commercially available BCA protein assay kit (Spectra Max190, Molecular Devices, USA). An enzymatic assay for SIRT1 activity was performed according to the manufacturer's instructions (Sigma-Aldrich). The plates were read with a fluorescent spectrophotometer (Spectra Max190, Molecular Devices) at 340 nm excitation and 430 nm emission.
6
Proliferation and Viability of HRMEC
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Proliferation of HRMEC was determined using a 5-bromo-2’-deoxyuridine (BrdU) ELISA in accordance to the manufactures recommendations (Roche, Mannheim, Germany). In brief, 1.2x104 cells/cm2 HRMEC were seeded onto a 96 well plate and incubated for 24 h to achieve complete adherence of the cells. After incubation with hr-GAL1 or 3 and BrdU labeling solution for 48 h, cells were fixed and incubated with anti-BrdU antibodies in accordance to the manufacturer’s instructions. Following the substrate reaction, the product formation was quantified by absorbance measurement at a wavelength of 450 nm and a reference at 690 nm on the SpectraMax 190 ELISA reader (Molecular Devices, San Jose, CA).
To analyze cell viability, a colorimetric dye reduction assay was conducted matching the manufacturer’s recommendations (WST-1; Roche). In brief, 1,5x104 cells/cm2 were seeded and incubated for 24 h. After treatment of HRMEC with hr-GAL1 or 3 for 72 h, WST-1 was added and incubated for additional 2 h. For readout, absorbance measurement at a wavelength of 450 nm and a reference at 690 nm was determined on a SpectraMax 190 ELISA reader (Molecular Devices).
To analyze cell viability, a colorimetric dye reduction assay was conducted matching the manufacturer’s recommendations (WST-1; Roche). In brief, 1,5x104 cells/cm2 were seeded and incubated for 24 h. After treatment of HRMEC with hr-GAL1 or 3 for 72 h, WST-1 was added and incubated for additional 2 h. For readout, absorbance measurement at a wavelength of 450 nm and a reference at 690 nm was determined on a SpectraMax 190 ELISA reader (Molecular Devices).
7
Polyphenol and Flavonoid Quantification
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Total polyphenol and flavonoid content were measured, as they are known to show a proportional correlation with antioxidant activity. Polyphenol content was determined using the Folin–Ciocalteu method. After roasting, 2 mL of 2% Na2CO3 solution was added to 100 μL of the sample extracted using 50% ethanol and left for 3 min. Then, 100 μL of 50% Folin–Ciocalteu reagent was added, and the mixture was left at room temperature for 30 min. The absorbance was measured at 750 nm using a UV-Spectrophotometer (SPECTRA MAX 190; Molecular Devices LLC, USA). Gallic acid (Sigma Aldrich, St. Louis, MO, USA) was used as the reference material, and the total polyphenol content was expressed as mg Gallic acid in 100 g of the sample. Total flavonoid content was determined by the following method. Then, 250 μL of sample was mixed with 1 mL of distilled water, 75 μL of 5% NaNO2, 150 μL of 10% AlCl3·6H2O, and 500 μL of 1 N NaOH. The mixture was allowed to react at room temperature for 10 min and the absorbance was measured at 510 nm using a UV-Spectrophotometer (SPECTRA MAX 190, Molecular Devices LLC, USA). Quercetin (Sigma-Aldrich, St. Louis, MO, USA) was used as the reference material, and the flavonoid content was expressed as mg Quercetin in 100 g of the sample.
8
Quantifying Protein and VEGF in BALF
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Total protein concentration in the BALF was determined using a bicinchoninic acid assay kit (P0010S, Beyotime biotechnology, Nantong, Jiangsu province, China) [10 (link)]. The optical densities were recorded using an enzyme mark instrument (SPECTRAMAX 190, Molecular Devices Corp., Sunnyvale, CA, USA) at 450 nm wavelength. A standard curve was thus generated to calculate the protein concentrations in BALF.
The VEGF level in BALF was determined using an enzyme-linked immunosorbent assay kit for mouse VEGF according to manufacturer’s instruction (MMV00, R&D Systems incorporation, Minneapolis, MN, USA) [11 (link)]. A specific monoclonal antibody was precoated onto a microplate before a sample was pipetted into the well. A polyclonal detection antibody was added subsequently prior to addition of the substrate solution to the well. The optical density developed in proportion to the amount of specific cytokine was recorded using an enzyme mark instrument (SPECTRAMAX 190, Molecular Devices Corporation, Sunnyvale, CA, USA) at 450 nm wavelength. A standard curve was then generated to calculate the concentrations of VEGF in BALF.
The VEGF level in BALF was determined using an enzyme-linked immunosorbent assay kit for mouse VEGF according to manufacturer’s instruction (MMV00, R&D Systems incorporation, Minneapolis, MN, USA) [11 (link)]. A specific monoclonal antibody was precoated onto a microplate before a sample was pipetted into the well. A polyclonal detection antibody was added subsequently prior to addition of the substrate solution to the well. The optical density developed in proportion to the amount of specific cytokine was recorded using an enzyme mark instrument (SPECTRAMAX 190, Molecular Devices Corporation, Sunnyvale, CA, USA) at 450 nm wavelength. A standard curve was then generated to calculate the concentrations of VEGF in BALF.
9
Measurement of SOD and Catalase Activities
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For the measurement of SOD activity, cells were suspended in cold HEPES buffer (20 mM HEPES pH 7.2, 1 mM EGTA, 210 mM mannitol, 70 mM sucrose), and the resulting cell lysates were centrifuged at 1500' g for 5 min at 4℃. SOD activity was measured using a SOD assay kit (Cayman Chemical Company, Ann Arbor, MI). In this assay, tetrazolium salt reacts with superoxide anion to produce formazan, which is detected at 450 nm with an ELISA microplate reader (SpectraMax190; Molecular Devices, Sunnyvale, CA).
For the measurement of catalase activity, cells were homogenized with cold phosphate buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA), and the resulting cell lysates were centrifuged at 10,000'g for 15 min at 4℃. Catalase activity was determined in supernatants by the reaction of catalase with methanol in the presence of an optimal concentration of H2O2. Formaldehyde production was determined using a catalase assay kit (Cayman Chemical Company) and was quantified by measuring absorbance at 540 nm using an ELISA microplate reader (SpectraMax190, Molecular Devices).
For the measurement of catalase activity, cells were homogenized with cold phosphate buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA), and the resulting cell lysates were centrifuged at 10,000'g for 15 min at 4℃. Catalase activity was determined in supernatants by the reaction of catalase with methanol in the presence of an optimal concentration of H2O2. Formaldehyde production was determined using a catalase assay kit (Cayman Chemical Company) and was quantified by measuring absorbance at 540 nm using an ELISA microplate reader (SpectraMax190, Molecular Devices).
10
In vitro α-Glucosidase Inhibition Assay
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In vitro activity on α-glucosidase was carried out according to the method described previously 19 , with some modifications. Briefly, to each 50 µl of each extract was added 50 µl of 0.1 M potassium phosphate buffer (pH 6.9) and 100 µl of 0.1 M potassium phosphate buffer (pH 6.9) containing α-glucosidase solution (0.5 U/ml) of Saccharomyces cerevisiae, the mixture was incubated at 37 °C for 30 min. Then, 50 µl of 5 mM solution of p-nitrophenyl-D-α-glucopyranoside (PNPG) in 0.1 M potassium phosphate buffer (pH 6.9) was added, and the mixture was incubated at 37 °C for 5 min. To stop the reaction, 50 µl of sodium carbonate (Na 2 CO 3 ) was added and incubated for 5 min. The absorbance was read at 405 nm in an ELISA reader (Spectra max 190, Molecular Devices). Before and after incubation, the change in absorbance of the sample (A 405 extract) and control (A 405 control) containing 50 µL of buffer solution instead of the extract was recorded in an ELISA reader (Spectra max 190, Molecular Devices). All the samples were processed in triplicate. The percentage of α-glucosidase inhibition was calculated using Eq. ( 2) below:
(2)
(2)
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