RNA-binding assays were conducted as described with minor adjustments 15 (link),18 (link). Luminescence data were collected using the β-Glo reagent (Promega) and measured with a 96-well synergy-2 plate reader (BioTek).
Synergy 2 96 well plate reader
Manufactured by Agilent Technologies
The Synergy 2 96-well plate reader is a multi-mode microplate reader designed for a variety of detection technologies. It features a xenon flash lamp and dual optics design to enable absorbance, fluorescence, and luminescence measurements.
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Lab products found in correlation
5 protocols using synergy 2 96 well plate reader
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RNA-Binding Assays with Luminescence
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RNA-Binding Assays with Luminescence
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Quantitative and Qualitative Endocytosis of Extracellular Vesicles
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EVs were fluorescently labeled using the ExoGlow exosome protein labeling kit (System Biosciences) as per the manufacturer’s protocols. For quantitative experiment, MSCs and BMMs were seeded onto 96 well tissue culture plates (10,000 MSCs/well, 20,000 BMMs/well) and incubated for overnight to facilitate cell attachment. The cells were then incubated with increasing numbers of fluorescently labeled EVs for 2 hours at 37°C, washed with PBS and fixed in neutral buffered 4% paraformaldehyde (PFA). The fluorescence from the endocytosed EVs was observed and quantified by using a BioTek Synergy 2 96 well plate reader equipped with the appropriate filter sets to measure green fluorescence. The results were plotted as mean (+/− SD) relative fluorescence intensity % increase (normalized to no EV group) as a function of dosage (n=6 per group).
For qualitative endocytosis experiments, 50,000 MSCs or BMMs were seeded onto cover glasses placed in 12 well or 24 well cell culture plates. Fluorescently labeled EVs (1.8x109 particles/well) were added and incubated for 2 hours. The cells were then washed with PBS, fixed in 4% PFA, permeabilized and counter stained using Alexa Fluor® 568 Phalloidin (1/2000, A12390, Invitrogen) antibody. The cover glasses were then mounted using mounting medium with DAPI (Vector Laboratories) and imaged using a Zeiss LSM 710 Meta confocal microscope.
For qualitative endocytosis experiments, 50,000 MSCs or BMMs were seeded onto cover glasses placed in 12 well or 24 well cell culture plates. Fluorescently labeled EVs (1.8x109 particles/well) were added and incubated for 2 hours. The cells were then washed with PBS, fixed in 4% PFA, permeabilized and counter stained using Alexa Fluor® 568 Phalloidin (1/2000, A12390, Invitrogen) antibody. The cover glasses were then mounted using mounting medium with DAPI (Vector Laboratories) and imaged using a Zeiss LSM 710 Meta confocal microscope.
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Measuring Hydrogen Peroxide Production
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Measuring Hydrogen Peroxide Production
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100 μM N-methyl-DL-DOPA or DL-DOPA, either with or without 15 μM FsqB, were incubated in 100 mM pH 7.0 potassium phosphate buffer at 25 °C in 100 μL total reaction volume in the dark in a 96-well plate with gentle shaking. After 1.5 h of incubation, 100 μL of hydrogen peroxide detection mixture (containing 50 mM potassium phosphate pH 7.0, 100 μM Amplex Red (Thermo Scientific), 0.2 units/mL HRP) was added and incubated at 25 °C in the dark for 10 minutes. Readings were performed on a BioTek Synergy 2 96-well plate reader using a 500±27 nm excitation filter and a 615±15 nm emission filter and normalized to 0 and 100 μM hydrogen peroxide standards. All conditions were performed in triplicate.
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