Intracellular ROS generation was detected using 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, St. Louis, MO, USA). Briefly, the cells were seeded in 96-well black plates(100 µl per well of culture medium) with a density of 5 × 104 /ml with 6 parallel wells in each group. Cells were stained with 10µM DCFH-DA at 37 °C in the dark for 15 min. Then cells were washed with serum-free F12K for three times, the level of ROS was determined using a fluorescence plate reader (Molecular Devices, San Jose, CA, USA) at 488/525nm. Mitochondrial ROS(mtROS) level was measured using MitoSOX Red (Invitrogen, Life Technologies, Carlsbad, CA, USA). Briefly, the cells were seeded in 96-well black plates with a density of 5 × 104 /ml with 6 parallel wells in each group. Cells were incubated with 5mmol/L MitoSOX Red probe for 10 min at 37 °C. The cells were washed twice with PBS, and red fluorescence was determined at 510/580nm using a fluorescence plate reader (Molecular Devices).
Fluorescence plate reader
Manufactured by Molecular Devices
Sourced in United States, United Kingdom
The Fluorescence plate reader is a core laboratory instrument designed to detect and quantify fluorescent signals in multi-well microplates. It measures the intensity of fluorescent light emitted from samples in each well, enabling researchers to analyze a wide range of fluorescence-based assays.
Automatically generated - may contain errors
Lab products found in correlation
H2dcfda, by Merck Group (4 mentions)
Griess reagent, by Promega (3 mentions)
Cas12a, by New England Biolabs (3 mentions)
Real time pcr detection system, by Bio-Rad (3 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (2 mentions)
Dcfh da, by Merck Group (2 mentions)
Ssdna reporter, by Sangon (2 mentions)
Mini beadbeater 8, by Biospec (2 mentions)
Sensolyte 520 mmp assay system, by AnaSpec (2 mentions)
Resazurin, by Merck Group (2 mentions)
Mda lysis buffer, by Merck Group (1 mentions)
Glutathione colorimetric detection kit, by Abcam (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Dcfda h2dcfda cellular ros assay kit, by Abcam (1 mentions)
Ab32117, by Abcam (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Rhod 2 am, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Ckx53sf, by Olympus (1 mentions)
Mitosox red probe, by Thermo Fisher Scientific (1 mentions)
56 protocols using fluorescence plate reader
1
Intracellular and Mitochondrial ROS Assay
2
Quantifying Lipid Peroxidation in Mouse Brain
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Mouse brain tissues were homogenized on ice using 300 μl of the MDA lysis buffer according to the manufacturer’s guidance (Sigma-Aldrich, St. Louis, MO) (with 3 μl of butylated hydroxytoluene [100X]), followed by centrifugation (13,000×g, 10 min) to remove insoluble materials. The supernatant (200 μl) from each homogenized sample was placed into a micro-centrifuge tube and incubated with 600 μl of thiobarbituric acid solution at 95 °C for 60 min. Cooled to RT in an ice bath for 10 min, 200 μl of mixtures was pipetted into a 96-well microplate and analyzed by using a fluorescence plate reader (Ex: 532 nm; Em: 553 nm) (Molecular Devices, Sunnyvale, CA).
3
Lymphoblastoid Cell Adhesion Assay
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TR-iBRB2 cells pretreated with the indicated drugs were plated at a density of 1.2×104 cells/well in flat clear-bottom black 96-well plates, and cultured to confluence overnight. Lymphoblastoid cells were harvested and suspended in RPMI 1640 with 10% FBS and then incubated with 2 mM C-AM for 30 min at 37 °C. After incubation, the cells were washed with PBS (1X; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) three times to remove the free C-AM. The suspension of the C-AM labeled cells (5×105 cells/well) was added to the monolayer of TR-iBRB2 cells for 90 min at 37 °C. After incubation, non-adherent cells were removed by washing with cell culture. C-AM fluorescence in cells was measured in a fluorescence plate reader (Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 490 nm and an emission wavelength of 530 nm [15 (link)].
4
Cell Viability Assessment by MTS Assay
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The cell-viability/-proliferation assay (MTS) was performed using One Solution Cell Proliferation Assay kit (CellTiter 96® AQueous One Solution, Promega, Madison, WI, USA), as per the manufacturer’s instruction. The results of MTS assay were obtained by measuring absorbance at 490 nm with a fluorescence-plate reader (Molecular Devices, Sunnyvale, CA, USA). All assays were performed in triplicate and experiments were repeated three times.
5
Quantifying Cellular Glutathione Levels
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To assay total cellular glutathione (GSH), tumor and normal cells were exposed to PL and N-acetyl-L-cysteine (3 mM, NAC, Sigma-Aldrich). Cells (1 × 106) were collected, centrifuged and lysed in 100 μL ice-cold lysis buffer for 10 min. The lysate was centrifuged for 10 min and the supernatant was used to assay GSH with a glutathione colorimetric detection kit (BioVision Inc., Milpitas, CA, USA). The total amount of GSH was measured using a fluorescence plate reader (Molecular Devices) at excitation (ex)/emission (em) = 380/460 nm. Quantification of glutathione disulfide (GSSG) was performed using a GSH/GSSG detection kit (ABcam, Cambridge, MA, USA). The drug-treated cells were collected in ice-cold buffer, homogenized and sonicated in icy water. GSH quencher (10 μL) was added and incubated for 10 min to quench GSH at room temperature. The samples were centrifuged and the supernatant was used to determine the GSSG concentration according to the manuscript protocol using a fluorescence plate reader. The change in GSSG levels in PL-treated samples compared to DMSO-treated control samples was expressed as the fold change.
6
Measuring ROS and Nitric Oxide in A549 and WI-38 Cells
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A549 or WI-38 cells (2 × 104 cells per well in a 24-well plate) were pretreated with BPE for 1 h and then stimulated with poly (I:C) (10 μg/mL) for 24 h. The supernatants of the cultured cells were collected, and the accumulated nitrite (NO2-, one of two primary stable and nonvolatile breakdown products of nitric oxide (NO)) was measured using Griess reagent (Promega, Madison, WI, USA). The intracellular accumulation of ROS was measured with DCFDA / H2DCFDA-Cellular ROS Assay Kit (ab113851, Abcam) according to the manufacturer's instructions. In brief, A549 or WI-38 cells were pretreated with BPE for 1 h, then stimulated with poly (I:C) (10 μg/mL) for 24 h, and then stained with 20 mM H2DCF-DA in PBS for 1 h at 37°C. DCF fluorescence intensity was measured at 485-nm excitation and 535-nm emission using a fluorescence plate reader (Molecular Devices, CA, USA).
7
Quantifying HDAC2 Activity in Stroke Samples
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HDAC activity in peri‐infarct cortical samples was measured according to the manufacturer's protocol (EMD Millipore). For HDAC2‐specific activity, immunoprecipitation with HDAC2 antibody was performed before the assay as described previously.21 Briefly, tissue lysates prepared in immunoprecipitation buffer (50 mmol/L Tris‐HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.5% NP‐40, pH 8.0, supplemented with 1 mmol/L phenylmethyl sulfonylfluoride (PMSF)) were incubated with 0.5 μL mouse anti‐HDAC2 (ab32117, Abcam), and 20 μL protein G‐Agarose (Sigma) overnight on a tube rotator at 4°C. Then beads were centrifuged at 5000g and washed 5 times in PBS. HDAC assay substrate was added to the beads and incubated at 30°C for 40 minutes. Finally, activator solution containing trichostatin A (HDAC inhibitor) was used to stop the reaction, and the supernatant was used for fluorescent measurement. Fluorescent was measured in 384 wells plate by excitation wavelength 360 nm and emission wavelength 450 nm using a fluorescence plate reader (Molecular Devices). HDAC activity was normalized to total protein levels determined by Bradford assay.
8
Quantifying Mitochondrial Calcium Levels
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Mitochondrial fractions (250 μg) were incubated at 37 °C with Rhod-2-AM (5 μM; Molecular Probes) for 1 h, followed by washing with Ca2+-free Locke’s solution (3–4 times). This reduced form of Rhod-2-AM is a colorless, non-fluorescent dye with a net positive charge, which promotes sequestration into the mitochondria, whereas dye oxidized in the mitochondria and cleaved AM ester are trapped inside the mitochondria. Fluorescence plate reader (Molecular Devices Inc.) was used to measure fluorescence with an excitation wavelength of 549 nm and emission wavelengths of 581 nm [13 (link),108 (link),109 (link),110 (link)].
9
Mitochondrial Membrane Potential Assay
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5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolycarbocyanine iodide dye (JC-1; Molecular Probes, Eugene, OR, USA) was used to assess mitochondrial transmembrane potential. This dye exists as a green-fluorescent monomer at low membrane potential but reversibly forms red-fluorescent “J-aggregates” at polarized mitochondrial potentials. Briefly, 250 μg aliquots of isolated mitochondrial protein from hypothalamic tissues were suspended in respiration buffer comprising 20 mM HEPES, 2.5 mM inorganic phosphates (pH 7.2), 250 mM sucrose, 2 mM MgCl2, and 10 mM succinate (5 mM glutamate and 2.5 mM maleate gave similar results in all paradigms) in a final volume of 200 μL. The energized mitochondria were then incubated with 10 μM JC-1 for 30 min at 37 °C, and fluorescence was measured using a fluorescence plate reader (Molecular Devices Inc., Sunnyvale, CA, USA). Mitochondrial polarization was measured by taking the emission ratio from 590 nm to 535 nm with excitation at 490 nm [13 (link),105 (link),106 (link),107 (link),108 (link)].
10
Quantification of Intracellular Compounds
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