Ultrospect 3100 pro spectrophotometer

Manufactured by Cytiva

The Ultrospect 3100 pro is a high-performance spectrophotometer designed for use in laboratory environments. It is capable of accurately measuring the absorption and transmission of light through samples across a wide range of wavelengths. The Ultrospect 3100 pro provides precise and reliable data for quantitative and qualitative analysis of various substances.

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Lab products found in correlation

Cell lysis buffer, by Cell Signaling Technology (2 mentions) Cytochrome c oxidase kit, by Merck Group (1 mentions) Atp bioluminescence assay kit, by Roche (1 mentions) Luminescence plate reader, by Molecular Devices (1 mentions)

5 protocols using ultrospect 3100 pro spectrophotometer

Respiratory Chain Enzyme Activity Assay

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Activity of key enzymes associated with the respiratory chain was
measured using homogenates of cerebral cortex including hippocampus as
previously described [22 (link)–24 (link)]. Briefly, sections of cerebral cortex
were homogenized in Cell Lysis buffer (Cell Signaling Technology). Homogenates
containing 50 μg of protein were used for complex I (NADH-ubiquinone
oxidoreductase) and IV (cytochrome c oxidase) assays. Complex I activity was
determined in 25 mM potassium buffer containing 3 M KCl, 1 M Tris-HCl, and 0.5 M
EDTA (pH 7.4). The change in absorbance was monitored at 340 nm wavelength every
20 s for 6 min using an Amersham Biosciences Ultrospect 3100 pro
spectrophotometer. Complex IV activity was determined by cytochrome c reduction
rate. Briefly, the protein samples were added to a cuvette containing 0.475 ml
of assay buffer (10 mM Tris-HCL, pH 7.0, and 120 mM KCl), and the reaction
volume was brought to 0.525 ml with enzyme dilution buffer (10 mM Tris-HCl, pH
7.0 and 250 mM sucrose). The reaction was initiated by the addition of
ferrocytochrome c substrate solution (0.22 mM) into the cuvette. The rate of
change in absorbance at 550 nm was recorded using a kinetic program with 5 s
delay and 10 s intervals for 6 readings on Amersham Biosciences Ultrospect 3100
pro spectrophotometer.

Akhter F., Chen D., Akhter A., Sosunov A.A., Chen A., McKhann G.M., Yan S.F, & Yan S.S. (2020). High Dietary Advanced Glycation End Products Impair Mitochondrial and Cognitive Function. Journal of Alzheimer's disease : JAD, 76(1), 165-178.

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Cytochrome c Oxidase Activity Assay

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The cytochrome c oxidase activities of mitochondrial fractions were measured with a cytochrome c oxidase kit (Sigma). Briefly, a suitable volume of mitochondria fraction or tissues and enzyme solution was added to 950 µl assay buffer. The reaction was initiated by the addition of 50 µl freshly prepared ferrocytochrome c substrate solution into the cuvette. Changes in OD values at 550 nm were recorded immediately using a kinetic program with 5 seconds’ delay, 10 seconds’ interval; a total of six readings were obtained utilizing an Amersham Biosciences Ultrospect 3100 pro spectrophotometer.

Du F., Yu Q., Yan S., Zhang Z., Vangavaragu J.R., Chen D., Yan S.F, & Yan S.S. (2021). Gain of PITRM1 peptidase in cortical neurons affords protection of mitochondrial and synaptic function in an advanced age mouse model of Alzheimer’s disease. Aging Cell, 20(5), e13368.

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Oxidative Stress Effects on Mitochondrial Complexes

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Briefly, cells (10⁶ cells/dish) were grown in 150-mm dishes until fully confluent. For measurement of complex I-IV enzymatic activities in response to oxidative stress, 0.75 mM of H₂O₂ was added to cells and incubated for 1, 6, 16, and 24 hours before collection. Cells were washed twice with pre-chilled PBS, and then harvested, centrifuged, and suspended in 100 μl of isolation buffer containing 225 mM D-mannitol, 75 mM sucrose, and 2 mM K₂HPO₄. A working solution of 25 mM potassium (K) buffer (3 M KCl, 1 M Tris–HCl, pH 7.4, and 0.5 M EDTA) was used for complex I, II, III, and IV assays as described [25 (link)]. An Ultrospect 3100 Pro-spectrophotometer (Amersham Biosciences, Piscataway, NJ) was used to measure the change in absorbance for all complexes. Background levels were measured in the absence of cell suspensions. All complex enzyme activities are expressed as nanomoles of substrate oxidized per mg⁻1 protein min⁻1 ml⁻1 (nmol/mg protein/min/ml).

Carlson E.A., Marquez R.T., Du F., Wang Y., Xu L, & Yan S.S. (2015). Overexpression of 17β-hydroxysteroid dehydrogenase type 10 increases pheochromocytoma cell growth and resistance to cell death. BMC Cancer, 15, 166.

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Mitochondrial Respiration Complex Assay

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Mitochondrial respiration complex activity was measured in hippocampi or in SK cell homogenates as previously described (37 (link),39 (link)). Briefly, hippocampi or cultured cells were homogenized and sonicated in the isolation buffer containing 250 mmol/L sucrose; 20 mmol/L HEPES, pH 7.2; and 1 mmol/L EDTA. NADH-ubiquinone oxidoreductase (COXI) enzyme activity was determined in 25 mmol/L potassium buffer containing KCl, Tris-HCl ,and EDTA (pH 7.4). The change in absorbance was monitored at 340 nm wavelength every 20 s for 6 min using an Amersham Biosciences Ultrospect 3100 pro spectrophotometer. For homogenized samples (50 μg protein), the oxidation of NADH was recorded for 3 min after the addition of 2 μg/mL antimycin, 5 mmol/L MgCl₂, 2 mmol/L KCN, and 65 μmol/L coenzymes Q1 to the assay mixture, and then 2 μg/mL rotenone was added to the mixture. The absorbance of samples was measured for another 3 min. Enzyme activities in complex II, complex III, complex IV, and citrate synthase activity were determined as previously described (37 (link)). We also determined ATP content in hippocampus using an ATP Bioluminescence Assay Kit (Roche) according to manufacturer’s instructions with a Luminescence plate reader (Molecular Devices) with an integration time of 10 s.

Huang S., Wang Y., Gan X., Fang D., Zhong C., Wu L., Hu G., Sosunov A.A., McKhann G.M., Yu H, & Yan S.S. (2014). Drp1-Mediated Mitochondrial Abnormalities Link to Synaptic Injury in Diabetes Model. Diabetes, 64(5), 1728-1742.

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Respiratory Chain Enzyme Activity Assay

Cited 1 time

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Activity of key enzymes associated with the respiratory chain was measured using homogenates of the cerebral cortex including hippocampus as previously described [26 (link)]. Briefly, cerebral cortices were homogenized in Cell Lysis buffer (Cell Signaling Technology). Protein extracts (50 μg) were used for complex I (COX I, NADH:ubiquinone oxidoreductase) and IV (COX IV, cytochrome c oxidase) assays. Complex I activity was determined by monitoring the change in absorbance at 340 nm every 20 s for 6 min. Complex IV activity was determined by cytochrome c reduction rate. Briefly, the protein samples were added to a cuvette containing 0.475 ml of assay buffer (10 mM Tris-HCL, pH 7.0, and 120 mM KCl), and the reaction volume was brought to 0.525 ml with enzyme dilution buffer (10 mM Tris-HCL, pH 7.0 and 250 mM sucrose). The reaction was initiated by the addition of ferrocytochrome c substrate solution (0.22 mM) into the cuvette. The rate of change in absorbance at 550 nm was recorded using a kinetic program with 5 s delay and 10 s intervals for 6 readings using an Amersham Biosciences Ultrospect 3100 pro spectrophotometer.

Akhter F., Chen D., Akhter A., Yan S.F, & Yan S.S. (2021). Age-dependent accumulation of dicarbonyls and advanced glycation endproducts (AGEs) associates with mitochondrial stress. Free radical biology & medicine, 164, 429-438.

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