Etheno-NAD cleaving activity was measured by a fluorimetric assay with etheno-NAD as the substrate [2 (link)]. Neutrophils or other immune cell types were suspended in Hank's balanced salt solution (HBSS) (in mM) (KCl 5.4, Na2HPO4 0.338, KH2PO4 0.44, NaHCO3 4.2, CaCl2 1.3, MgCl2 0.5, MgSO4 0.6, NaCl 137, D-glucose 5.6) and seeded to 96 well-plate at a cell density of 1×107 cells/ml. Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD, which causes a 10-fold fluorescence enhancement, was continuously followed at 37℃ using Spectramax M2/e fluorescence microplate reader (Molecular Devices) at an excitation of 300 nm and an emission wave length of 410 nm.
Spectramax m2 e fluorescence microplate reader
Manufactured by Molecular Devices
The SpectraMax M2/e fluorescence microplate reader is a versatile instrument designed for accurate and sensitive fluorescence measurements in microplate format. It offers a high-performance optics system and a wide dynamic range to support a variety of fluorescence-based applications.
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Lab products found in correlation
2 protocols using spectramax m2 e fluorescence microplate reader
1
Fluorimetric Assay for Etheno-NAD Cleavage
2
Neutrophil-derived EV Isolation and Analysis
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Neutrophils (5 × 106 cells) were stained with calcein-AM (20 μg/mL, Merck Millipore) and stimulated with various stimulators: fMLP (1 μM), LPS (1 μg/mL), E. coli (1 × 106 cells), S. aureus (1× 106 cells), C5a (50 ng/mL), S100B (100 ng/mL), HMGB1 (100 ng/mL), TNF-α (50 ng/mL), IFN-γ (100 ng/mL), TGF-β (20 ng/mL), IL-4 (20 ng/mL), PMA (100 μg/mL), and L-NAME (10 μM). Then neutrophil-derived EVs were isolated and the fluorescence were measured using Spectramax M2/e fluorescence microplate reader (Molecular Devices).
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