A high throughput endonuclease assay was performed as described previously.15 The endonuclease assay used a single-stranded DNA probe with the sequence (6-FAM)TGGCAATATCAGCTCCACA(MGBNFQ) (Applied Biosystems). Reaction buffer contained 50 mM Tris pH 7.5, 50 mM sodium chloride, 1 mM DTT, 5 mM magnesium chloride, 0.5 mM manganese sulfate, and 1 mM CHAPS. DNA probe (50 nM), endonuclease (25 nM), and compound were mixed on ice and then placed in a Varioskan Fluorometer (Thermo Scientific) preincubated at 37 ˚C to detect fluorescence with an excitation of 488 nm and emission of 518 nm. Fluorescence is measured at several time points (5, 60, and 90 minutes) and activity/inhibition is calculated based on the change in fluorescence over time using Prism Graphpad (6.0b) non-linear regression analysis. Ligand efficiency was calculated using the following equation: LE (in kcal/(mol·NHA)) = −RTln(IC50)/NHA.
Varioskan fluorometer
Manufactured by Thermo Fisher Scientific
The Varioskan Fluorometer is a multi-mode microplate reader designed for fluorescence-based assays. It can measure fluorescence intensity, time-resolved fluorescence, and fluorescence polarization. The Varioskan Fluorometer is capable of performing a wide range of fluorescence-based applications in a variety of sample formats.
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Lab products found in correlation
2 protocols using varioskan fluorometer
1
Fluorescence-based Endonuclease Inhibition Assay
2
Quantification of ROS and Nitric Oxide
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Quantification of ROS and nitric oxide (NO) production was performed using 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA; ref C2938) and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM; ref D23844) probes, respectively, following supplier’s recommendations (Molecular Probes, ThermoFisher Scientific). Briefly, MSCs were plated at 50,000/cm2 in six-well plates in proliferative medium and incubated with 50 µM of DCFDA or 10 µM of DAF2 at 37°C for 60 min. Probes were then washed out with PBS. Media containing 5% human serum (SAB, SAB400, SAB1000, SABH2O2, or PS) were added for 24 h. Fluorescence was read at different time points using a Varioskan fluorometer (ThermoFisher Scientific) at 495 nm for excitation and 515 nm emission. Results were expressed as the relative fluorescence unit (RFU) or gene expression fold change ± SEM and normalized to 1 for MSCs cultured in control SAB.
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