The in vitro antioxidant capacity of SOD-Like Supreme was determined by the ORAC assay. Briefly, an aliquot of 40 μL of SOD-Like Supreme, Trolox or ascorbic acid (at final concentrations of 0.0002, 0.001, 0.002 mg/mL in 50% v/v methanol in 75 mM potassium phosphate buffer, pH 7.4) was added to 120 μL of fluorescein solution (0.167 μM in potassium phosphate buffer), and the reaction mixture was incubated at 37 °C for 5 min. A 40 μL aliquot of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH, 80 mM in potassium phosphate buffer) was rapidly added to the reaction mixture and the fluorescence intensity (with excitation and emission wavelengths of 485 and 520 nm, respectively) was recorded every minute for 80 min using a Victor V3 Multi-label Counter (Perkin-Elmer, Wellesley, MA, USA). A blank containing the vehicle (i.e., 50% v/v methanol in 75 mM potassium phosphate buffer) was also measured. A fluorescence decay curve was constructed by plotting the % initial fluorescence intensity versus time, and the area under curve (AUC) was determined. An ORAC curve of the sample was then obtained by plotting the value of AUCsample − AUCblank against the concentration of the sample. The ORAC value was estimated by comparing the slope of the ORAC curve of SOD-Like Supreme or ascorbic acid to that of Trolox and expressed as Trolox equivalents.
Victor v 3 multilabel counter
Manufactured by PerkinElmer
Sourced in United States
The Victor V 3 multilabel counter is a versatile instrument designed for the detection and quantification of various biochemical and cell-based assays. It is capable of measuring absorbance, fluorescence, and luminescence in microplates. The instrument provides high sensitivity and wide dynamic range to support a broad range of applications.
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Lab products found in correlation
3 protocols using victor v 3 multilabel counter
1
ORAC Assay for Antioxidant Capacity
2
Cytotoxicity Assessment via Metabolic Activity and Membrane Integrity
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Cell viability was measured at the end of the exposure period essentially as described by Schreer et al. (2005) by use of the two probes Alamar blue (AB) and 5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester (CFDA-AM) for measuring the metabolic activity (AB) and membrane integrity (CFDA-AM). The probes are commonly used in combination to assess cytotoxicity.
CFDA-AM is hydrolysed to the fluorescent 5-carboxyfluorescein (CF) by unspecific esterases (Schreer et al 2005) which is negatively correlated with cellular damage (Schirmer et al., 1997) .
After 96 h of exposure, exposure media was removed and cells were incubated in tris buffer (50 mM, pH 7.5, 100 µl per well) containing 5% AB and 4 µM CFDA-AM. After 30 min incubation in the dark on an orbital shaker (100 rpm), the fluorescence was read using Victor V 3 multilabel counter (Perkin Elmer, Waltham, MA, USA) with wavelength pairs of excitation and emission of 530-590 (AB) and 485-530 (CFDA-AM). The results were normalised between the negative control (solvent, DMSO = 100% viability) and positive control (CuSO4 10 mM = 0% viability).
CFDA-AM is hydrolysed to the fluorescent 5-carboxyfluorescein (CF) by unspecific esterases (Schreer et al 2005) which is negatively correlated with cellular damage (Schirmer et al., 1997) .
After 96 h of exposure, exposure media was removed and cells were incubated in tris buffer (50 mM, pH 7.5, 100 µl per well) containing 5% AB and 4 µM CFDA-AM. After 30 min incubation in the dark on an orbital shaker (100 rpm), the fluorescence was read using Victor V 3 multilabel counter (Perkin Elmer, Waltham, MA, USA) with wavelength pairs of excitation and emission of 530-590 (AB) and 485-530 (CFDA-AM). The results were normalised between the negative control (solvent, DMSO = 100% viability) and positive control (CuSO4 10 mM = 0% viability).
3
Evaluating Cell Viability and Membrane Integrity
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At the end of the 96h exposure period, metabolic activity and membrane integrity were determined essentially as described by Schreer et al. (2005) using the two probes Alamar blue (AB) and 5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester (CFDA-AM), respectively.
The growth media was removed from the wells before the cells were incubated in 100 µl tris buffer (5 mM, pH 7.5) containing 5% AB and 4 µM CFDA-AM. Fluorescence was read after 30 min of incubation on an orbital shaker (100 rpm) in the dark (room temperature) at wavelength pairs of excitation and emission of 530-590 nm (AB) and 485-530 nm (CFDA-AM) using a Victor V 3 multilabel counter (PerkinElmer, Waltham, MA, USA). The results were normalized to the DMSO control (100% viability) and the highest concentration of CuSO4 (10 mM) causing 100% cell death (0% viability).
The growth media was removed from the wells before the cells were incubated in 100 µl tris buffer (5 mM, pH 7.5) containing 5% AB and 4 µM CFDA-AM. Fluorescence was read after 30 min of incubation on an orbital shaker (100 rpm) in the dark (room temperature) at wavelength pairs of excitation and emission of 530-590 nm (AB) and 485-530 nm (CFDA-AM) using a Victor V 3 multilabel counter (PerkinElmer, Waltham, MA, USA). The results were normalized to the DMSO control (100% viability) and the highest concentration of CuSO4 (10 mM) causing 100% cell death (0% viability).
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