Lacrimal pieces were incubated for 40 minutes in KRB-HEPES containing 4% BSA at 37°C in the presence of the agonists: carbachol, phenylephrine, ATP, and 2MeSATP. To terminate the incubation, pieces were pelleted by centrifugation, and the supernatant was collected. The pellet was homogenized in 10 mM Tris-HCl (pH 7.5). Peroxidase activity, an index of protein secretion, was measured in duplicate in both the supernatant and the pellet. Peroxidase was measured using Amplex Red reagent (Invitrogen), which, when oxidized by peroxidase in the presence of hydrogen peroxide, produces a highly fluorescent molecule. The amount of fluorescence in the supernatant and pellet was quantified using a fluorescence microplate reader (model FL600; BioTek) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Peroxidase was expressed as a percentage of peroxidase secreted into the media (supernatant) compared with total peroxidase present in the cells before stimulation (pellet plus supernatant). Data were expressed as fold increase over basal, which was set to 1.
Model fl600
Manufactured by Agilent Technologies
The Agilent Model FL600 is a fluorescence spectrometer designed for laboratory applications. It provides users with the ability to measure the fluorescence properties of samples. The instrument is capable of acquiring emission and excitation spectra.
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3 protocols using model fl600
1
Lacrimal Peroxidase Secretion Assay
2
Caspase-3 and Caspase-9 Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Caspase-3 and caspase-9 activities were detected by Caspase-3 Fluorescent Assay Kits and Caspase-9 Fluorescence assay kits (both from Clontech). The above treated cells were harvested and lysed with cell lysis buffer for 10 min. After centrifuge, 50 μl of 2 Reaction Buffer/DTT Mix and 5 μl specific substrate of DEVD-AFC (for caspase-3) or LEHD-AMC (for caspase 9) were added to the supernatants for further incubation at 37°C. One hour later, caspase activities were detected by a fluorometer (model FL600, Bio-Tek Instruments).
3
Lacrimal Peroxidase Secretion Assay
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