Fluorescent plate reader

Manufactured by Agilent Technologies

Sourced in United States

The Fluorescent Plate Reader is a versatile instrument used for quantitative analysis of fluorescent samples in a multiwell plate format. It accurately measures the fluorescence intensity of samples, providing data on various fluorescent properties.

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Lab products found in correlation

Dcfh da, by Merck Group (4 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Filipin, by Merck Group (1 mentions) Carboxy h2dcfda, by Thermo Fisher Scientific (1 mentions) Picogreen dsdna quantitation kit, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Resazurin salt, by Merck Group (1 mentions) Bovine alp, by Merck Group (1 mentions) Pnpp alkaline phosphatase assay kit, by Merck Group (1 mentions) Cutsmart buffer, by New England Biolabs (1 mentions) Murine rnase inhibitor, by New England Biolabs (1 mentions) Synergy htx multi mode reader, by Agilent Technologies (1 mentions) Human albumin elisa kit, by Thermo Fisher Scientific (1 mentions) Ros assay kit, by Beyotime (1 mentions) Alamar blue, by Agilent Technologies (1 mentions) Alamarblue reagent, by Thermo Fisher Scientific (1 mentions) Jc 1 mitochondrial membrane potential assay kit, by Cayman Chemical (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions) Du800 spectrophotometer, by Beckman Coulter (1 mentions)

Close spelling variants of this product

Synergy HT fluorescent plate reader Fluorescent microtiter plate reader Gemini fluorescent plate reader FL600 fluorescent plate reader Biotek synergy NEO2 fluorescent plate reader Synergy H1 fluorescent plate reader Synergy Mx fluorescent plate reader FLx800 fluorescent plate reader Synergy 2 fluorescent plate reader Fluorescent reader

26 protocols using fluorescent plate reader

Measuring Cellular H2O2 Production

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HEK293T were cultured in media Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). One day after seeding in 60 mm dish, 6 µg of human NOX4 WT and mutant (P473H, N129S, and T555S) plasmids were transfected into HEK293T cells using lipofectamine 2000 as per manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Forty-eight hrs after transfection, cells were harvested in Krebs-Ringer buffer and mixed with Amplex Red reaction buffer as previously described [9] (link). Each sample was treated with or without PEG-catalase (100 U/mL). The samples were incubated at 37°C for 1 h in the dark, and then centrifuged at 2,500g for 5 min. The supernatants were loaded to a 96 well plate, and then measured using a BioTek fluorescent plate reader at 530 nm excitation and 590 nm emission respectively. Freshly prepared H₂O₂ standards were used to calculate the amount of H₂O₂ produced by the samples.

Siu K.L., Li Q., Zhang Y., Guo J., Youn J.Y., Du J, & Cai H. (2016). NOX isoforms in the development of abdominal aortic aneurysm. Redox Biology, 11, 118-125.

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Measuring Intestinal Permeability via FITC-Dextran

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Gut leakiness was quantified by measuring blood levels of a 4kDa FITC-dextran administered by gavage as described (Aube et al., 2006 (link)). Mice were fasted for 2 hours then gavaged with 22 mg/mL FITC-dextran and blood was collected via cardiac puncture five hours later. Dextran levels in the serum were quantified by reading the fluorescence (excitation 480nm, emission 520 nm) via fluorescent plate reader (Bio-Tek).

Manocha G.D., Floden A.M., Miller N.M., Smith A.J., Nagamoto-Combs K., Saito T., Saido T.C, & Combs C.K. (2019). Temporal Progression of Alzheimer’s disease in Brains and Intestines of Transgenic Mice. Neurobiology of aging, 81, 166-176.

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Intracellular ROS Detection in HaCaT Cells

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For the present study, 1.5 × 10⁴ HaCaT cells were seeded in 96-well plates and incubated at 37 °C for 24 h. Subsequently, cells were pre-treated with samples (HPBCD, 734THIF, and 7HP) for 6 h before reaction with 20 μM dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma, Tokyo, Japan) solution, for detection of intracellular ROS. After 30 min, cells were incubated with 50 μg/cm² PM for an hour, washed twice with PBS, and soaked in PBS for detecting relative fluorescence intensity at excitation and emission wavelengths of 485 nm and 528 nm by fluorescent plate reader (BioTek, Winooski, VT, USA).

Huang P.H., Hu S.C., Yen F.L, & Tseng C.H. (2019). Improvement of Skin Penetration, Antipollutant Activity and Skin Hydration of 7,3′,4′-Trihydroxyisoflavone Cyclodextrin Inclusion Complex. Pharmaceutics, 11(8), 399.

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Membrane Sterol Staining Protocol

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Staining of membrane sterols was performed as previously described (36 (link), 62 (link)). Briefly, log-phase cultures grown in YPD were stained for 5 min with 5 μg/mL of filipin (Sigma) and washed with PBS. After washing cells were immediately visualized with an inverted Zeiss microscope. Fluorescence was calculated using Cell Profiler software. Additionally, fluorescent levels were quantified with a fluorescent plate reader (Bio-Tek). Fluorescence was measured at the range of 360 to 470 nm wavelength and normalized by OD₆₀₀.

Winski C.J., Qian Y., Mobashery S, & Santiago-Tirado F.H. (2022). An Atypical ABC Transporter Is Involved in Antifungal Resistance and Host Interactions in the Pathogenic Fungus Cryptococcus neoformans. mBio, 13(4), e01539-22.

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Quantifying Macrophage Oxidative Stress

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Macrophages were seeded in 48-well plates (5.3 x 10⁴ cell/cm²) for 24 hours prior to pretreatment with 10 μM dose of the non-fluorescent, membrane-permeable dye 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate(carboxy-H2DCFDA, ThermoScientific, Grand Island, NY) at 37°C for 20 min. Esterases in the cells convert carboxy-H2DCFDA to the charged form to increase intracellular retention. Cells were then washed with PBS before treatment with short and long fibers for 24-hours as detailed in the inflammatory biomolecule and cytotoxicity section above. carboxy-H2DCFDA is chemically reduced by intracellular reactive oxygen species (ROS) to become fluorescent. Cell fluorescence was detected using a fluorescent plate reader (Biotek, Winooski, VT)

Padmore T., Stark C., Turkevich L.A, & Champion J.A. (2016). Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages. Biochimica et biophysica acta, 1861(2), 58-67.

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Quantifying Cell Infiltration in Fibers

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DNA content of seeded cells on the fibers at various densities was quantified using a PicoGreen dsDNA quantitation kit (ThermoFisher Scientific). Cells were lysed by placing whole fibers in 500 μl of digestion buffer (10 mM Tris, 1 mM EDTA, 0.1% Triton X-100, and 0.1 mg/mL proteinase K) before incubation with PicoGreen and reading with a fluorescent plate reader (BioTek, Winooski, VT, USA) at excitation 485 nm and emission 530 nm. Cellular infiltration into the fiber interior was assessed using 10 μm-thick cryosections and staining for 30 min with DAPI (Sigma-Aldrich).

Gilbert-Honick J., Ginn B., Zhang Y., Salehi S., Wagner K.R., Mao H.Q, & Grayson W.L. (2018). Adipose-derived Stem/Stromal Cells on Electrospun Fibrin Microfiber Bundles Enable Moderate Muscle Reconstruction in a Volumetric Muscle Loss Model. Cell Transplantation, 27(11), 1644-1656.

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Metabolic Activity Quantification of AF Cells in PCL/PLLA Scaffolds

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Alamar blue was used to measure the metabolic activity of AF cells at 1 and 7 days on the PCL/PLLA 50:50 scaffolds (n = 3). Resazurin salt (Sigma) was dissolved in PBS at a concentration of 0.125 mg/mL and filter-sterilized to create a stock Alamar blue solution. A 1:9 dilution of stock solution was added to fresh media in each well and incubated for 4 h. For each sample, 200 μL was transferred (in triplicate) to a 96-well plate and read by a fluorescent plate reader (Biotek) at 530 nm excitation and 590 nm emission.

Shamsah A.H., Cartmell S.H., Richardson S.M, & Bosworth L.A. (2019). Mimicking the Annulus Fibrosus Using Electrospun Polyester Blended Scaffolds. Nanomaterials, 9(4), 537.

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Oxidative Stress Evaluation of Particulate Matter

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PM (Standard Reference Material, SRM^® 1649b) were purchased from the National Institute of Standards and Technology. This product was collected in 1976 and 1977 in Washington, DC Made. A total of 10 mg/mL of PM were suspended in PBS and then sonicated for 10 min before use. A total of 1 × 10⁴ HaCaT keratinocytes were cultured in 96-well plates for 24 h under 37 °C and 5% CO₂ conditions. Cells were treated with different concentrations of OA in DMSO, OA in PBS, and OAnf in PBS for 24 h, respectively. Then, they were incubated with 20 μM dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma, Tokyo, Japan) solution for 30 min. Next, 50 μg/cm² PM was added into each well and incubated for 1 h. After that, cells were washed twice with PBS, and the fluorescence intensity of each sample was analyzed using the fluorescent plate reader (excitation: 485 nm; emission: 528 nm) (BioTek, Winooski, VT, USA). The following equation was used to calculate the inhibition percentage of ROS production:

ROS production (%) = \frac{{OD}_{sample}}{{OD}_{control}} \times 100 %

Fu H., Yen F.L., Huang P.H., Yang C.Y, & Yen C.H. (2021). Oleanolic Acid Nanofibers Attenuated Particulate Matter-Induced Oxidative Stress in Keratinocytes. Antioxidants, 10(9), 1411.

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Cell Retrieval and ALP Activity Assay

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Cell-laden microfibers
after 7 days in culture were washed with 0.9% NaCl, soaked in 50 mM
EDTA and 0.2 mg/mL collagenase solution in 0.9% NaCl for 10 min at
room temperature and then incubated in trypsin solution for 10 min
to retrieve cells from the microfibers. The obtained cells were washed
in 0.9% NaCl, counted, and lysed for 30 min in a lysis buffer (0.1%
Triton X-100, 50 mM Tris-HCl) at 4 °C. The ALP activity was measured
with a pNPP Alkaline Phosphatase Assay Kit (Sigma) according to the
user’s manual. Bovine ALP (Sigma) was adopted to create a standard
curve. Absorbance determined at 405 nm was read by a fluorescent plate
reader (BioTek, USA) and normalized to cell counts.

Wei D., Charlton L., Glidle A., Qi N., Dobson P.S., Dalby M.J., Fan H, & Yin H. (2021). Dynamically Modulated Core–Shell Microfibers to Study the Effect of Depth Sensing of Matrix Stiffness on Stem Cell Fate. ACS Applied Materials & Interfaces, 13(32), 37997-38006.

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Quantifying Microglial Aβ Peptide Phagocytosis

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Peptide phagocytosis was quantitated by measuring the uptake of FITC-conjugated Aβ 1–42 (rPeptide, Athens, GA). The peptide was fibrillized according to manufacturer protocol and as previously described (Floden and Combs, 2006 (link)). Briefly, the peptide was dissolved in 2mM NaOH at 1mg/mL, sonicated for 30 seconds and diluted with 10× PBS and water to a final concentration of 50μM Aβ. The peptide was allowed to fibrillize 24hr at 37 degree C before use. Cultured microglia were incubated with Aβ 1–42 fibril aggregates or FITC-E. coli bioparticles (positive control, 0.125mg/ml) in 96-well plates for 6 hours. To quench the FITC signal from extracellular peptide or bioparticles, medium was removed and the cells were rinsed with 0.25mg/ml trypan blue in PBS. Application of trypan serves to quench any remaining FITC signal on the plate as well as any bound to the external leaflet of the plasmalemma. Intracellular fluorescence was read (480 nm excitation and 520 nm emission) via fluorescent plate reader (Bio-Tek, Winooski, VT).

Puig K.L., Kulas J.A., Franklin W., Rakoczy S.G., Taglialatela G., Brown-Borg H.M, & Combs C.K. (2016). The Ames Dwarf Mutation Attenuates Alzheimer’s Disease Phenotype of APP/PS1 mice. Neurobiology of aging, 40, 22-40.

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