Caspase activity was measured from all experimental cardiac tissues using ApoAlert caspase-3 and –9 Fluorescent Assay Kit (Clontech Laboratories, CA) following manufacturer’s protocol [17] (link). Briefly, tissue samples were homogenized in chilled protein extraction buffer. Then, 50 µl of 2X Reaction Buffer/DTT mix and 1 µl of Caspase-3 Inhibitor DEVD-CHO (for negative control) or 1 µl of DMSO (for other samples) was added to 50 µl of supernatant obtained from each sample. After incubation on ice for 30 min 5 µl of 1 mM Caspase-3 Substrate (DEVD-AFC; 50 µM final conc.) was added to each tube and incubated at 37°C for 1 hr. Fluorescence was measured at 400 nm excitation and 505 nm emission wavelengths (Varioskan Multimode Reader, Thermo Fisher, IL). For Caspase-9 activity, 5 µl of Caspase-9 Substrate (LEHD-AMC; 50 µM final conc.) was added to each tube and after incubation for 1 hr, fluorescence was measured at 380 nm excitation and 460 nm emission wavelength.
Varioskan multimode reader
Manufactured by Thermo Fisher Scientific
Sourced in France
The Varioskan Multimode Reader is a versatile laboratory instrument designed for a wide range of applications. It can perform absorbance, fluorescence, and luminescence measurements to support various research and analytical needs.
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Lab products found in correlation
Bicinchoninic acid (bca), by Merck Group (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
8 protocols using varioskan multimode reader
1
Quantifying Caspase Activity in Cardiac Tissues
2
Quantifying Redox Status via GSH Levels
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Oxidative stress was assessed by measuring changes in reduced glutathione (GSH) cellular levels using the 2,3-naphthalenedialdehyde (NDA) probe. Cells were seeded into 24-well culture plates at a density of 5 × 105 cells/well, differentiated into macrophages and incubated for 4 h with increasing concentrations of CDs. At the end of the CD exposure, cells were washed with 5 mM EDTA, 40 mM NaH2PO4, 110 mM Na2HPO4 (pH 7.4), and lysed with 0.1% Triton X100®. Then, proteins were denatured and precipitated (0.1 M hydrochloric acid, 50% sulfosalicylic acid) before sample centrifugation (10,000 g, 15 min, 4 °C). Cell lysates were then incubated with the NDA probe for 25 min at 4 °C, before fluorescence measurement (λex = 485 nm; λem = 528 nm) using a Varioskan multimode reader (Thermo Scientific, Illkirch-Graffenstaden, France). A calibration curve was used to calculate the amount of reduced GSH in the samples. This amount was then expressed in nmol of GSH per mg of protein. To do so, protein concentration in samples was determined using the bicinchoninic assay (BCA, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.
3
Characterization of Carbon Dots
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The hydrodynamic diameter of CDs was measured by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Paris, France) and calculated from the number distribution graph. Zeta potential was measured by DLS as well and calculated with the Smoluchowski’s equation. All measurements were performed in triplicate on fresh samples (1.0 mg/mL in 1.5 mM NaCl pH 7.4) at 25 °C. The surface charge density of CDs was determined by means of polyelectrolyte titration as described elsewhere [39 (link)]. In brief, the electrokinetic charge (Qek) of the CDs was determined by monitoring zeta potential variation in the sample (1.0 mg/mL, NaCl 1.5 mM pH 7.4) alongside spiking with a solution of poly(acrylic acid) (PAA, MW ± 1800 Da, NaCl 1.5 mM pH 7.4). Qek was calculated from the amount of PAA required to bring ζ down to zero. The data were expressed in µmol of amine per mg of material. Optical properties of the CDs were characterized by carrying out UV-visible and fluorescence measurements on CD samples (1.0 mg/mL in 1.5 mM NaCl pH 7.4) using a Varioskan multimode reader (Thermo Scientific, Illkirch-Graffenstaden, France).
4
Plasma Antioxidant and Oxidant Levels
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The total antioxidant levels of the plasma samples were studied using the method developed by Erel [8 (link)]. The test is based on the degradation of the blue-green color formed by ABTS (2,2’-azinobis-3-ethylebenzothiazoline-6-sulfonate) radical with antioxidants in the sample. ABTS is incubated with a peroxidase containing myoglobin (HX-Fe+) and H2O2 to form the ABTS+ radical. The resultant ferryl myoglobin reacts with ABTS to form the ABTS+ radical, it is blue-green in color. This formed color is inhibited according to the ratio of antioxidants in a sample and measured with a Varioskan Multimode Reader (Thermo Scientific, USA) at 600 nm. Ascorbic acid was used as a standard in the calculation of TAS.
The plasma total oxidant levels were determined using the method developed by Erel [9 (link)]. The total oxidant test is depending on the oxidation of ferric ion to ferrous ion in the availability of diverse oxidant species. To make it ironic, the oxidants oxidize the iron-a-dianisidine complex. The produced ferric ion makes a colored complex of xylenol orange-ferric ion. In a sample, the amount of oxidant will be associated with the intensity of color. This color-changing was measured with a Varioskan Multimode Reader at a wavelength of 530 nm. As a standard for TOS calculation, H2O2 was used. The oxidative stress index (OSI) was calculated as TOS/TAS.
The plasma total oxidant levels were determined using the method developed by Erel [9 (link)]. The total oxidant test is depending on the oxidation of ferric ion to ferrous ion in the availability of diverse oxidant species. To make it ironic, the oxidants oxidize the iron-a-dianisidine complex. The produced ferric ion makes a colored complex of xylenol orange-ferric ion. In a sample, the amount of oxidant will be associated with the intensity of color. This color-changing was measured with a Varioskan Multimode Reader at a wavelength of 530 nm. As a standard for TOS calculation, H2O2 was used. The oxidative stress index (OSI) was calculated as TOS/TAS.
5
Cell Viability Assessment in Prostate Cancer Cell Lines
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LNCaP, PC3 and DU145 cells (0.1 × 104) were seeded in a 96 well plate. Cells were transfected with FRG1 expression and silencing vectors along with their respective controls. Transfected cells were grown for 96 h. Afterwards culture medium was replaced with 100 μl fresh medium and, 20 μl of Cell titer 96 AQueous one solution reagent (Promega, USA) was added. Plates were incubated at 37 °C for 1 h. Absorbance was recorded at 490 nm wavelength using Varioskan multimode reader (Thermo Fisher Scientific).
6
Evaluating IRF3 and NF-κB Signaling Pathways
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HEK293 cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. IRF3 luciferase reporter gene assays were performed by transfection of constructs pCMV-MAVS, pTATA-luc-4x-IRF-3 (0.15 mg per 24-well), which contains the luciferase reporter gene under the control of four copies of the IRF-3 binding PRDI/III motif of the IFN-β promoter [35] (link) along with presence or absence of pcD-NSP1. For dual luciferase NFκB reporter assay, HEK293 cells were cotransfected with NFκB–luc (containing IL8 promoter), pRL-TK, pCMV-MAVS and pcD-NSP1 or pcDNA. 24 hour post transfection, the luciferase activity was measured according to the manufacturer’s protocol (Promega) using a luminometer (Varioskan multimode reader; Thermo Fisher).
7
Transfection and Cell Viability Assay
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HT29 cells were transfected with control miRNA or mimic or inhibitor of miR-99b and let-7g or miRNA cocktail or small-molecule inhibitors STO-609, LY294002, and 3-MA in 96 well plates. At 48 h post-transfection, cells were treated with 20 μl of the reagent solution in the 100 μl serum-free medium for 4 h at 37 °C in a humidified 5% CO2 atmosphere, as described by Cell titer 96® Aqueous One Solution Cell Proliferation Assay kit (Promega: G3581). The cell plate was measured spectrophotometrically at 490 nm in Varioskan Multimode Reader (Thermo Fisher Scientific). The absorbance was directly proportional to the number of living cells in culture. The percent viability was calculated considering 100% viability for mock control at similar endpoints.
8
Quantifying Ventricular Collagen Concentration
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Hydroxyproline assay was performed to measure ventricular collagen concentration both for in vivo experimental tissues as well as fibroblast culture supernatant (24 h treatment) [29] (link). Briefly, the tissue samples and fibroblast culture supernatants were subjected to acid digestion followed by vacuum drying. After resuspension in citrate acetate buffer, the samples were incubated with isopropyl alcohol, chloramine T, and Ehrlich’s reagent at 25°C for 18 h, and intensity of the red color was measured at 558 nm using Varioskan Multimode Reader (Thermo Fisher, IL). With the help of a standard curve, hydroxyproline content in the unknown samples was calculated. The amount of collagen was calculated by multiplying hydroxyproline content by a factor of 8.2.
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