Mitochondrial complex IV, also known as cytochrome C oxidase, is one of the major enzymes in the mitochondrial respiratory chain. It oxidizes the reduced cytochrome C converted from mitochondrial complex III to oxidized cytochrome C and consumes oxygen to generate water. Mitochondrial complex IV can catalyze the oxidation of reduced cytochrome C to oxidized cytochrome C, which has an absorption wavelength at 550 nm. Therefore, the activity of mitochondrial complex IV can be quantified by measure the change OD value at 550 nm.
Mitochondrial complex V is also known as F0F1-ATP synthase. ATP is hydrolyzed by F0F1-ATP synthase to produce ADP, and ADP converts NADH into oxidized coenzyme I (NAD) after enzyme conversion reaction. The activity of mitochondrial complex V can be quantified by measure the change OD value at 340 nm.
The differential centrifugation method is the most commonly used method for separating cell mitochondria. Usually, the first step is to prepare the cell homogenate, then perform low-speed centrifugation to remove the cell nucleus, cell debris, and undamaged cells, and finally perform high-speed centrifugation to precipitate the mitochondria.
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Abnormal accumulation of cellular cuprous in cells induces the oligomerization of key components of the lipoic acid-modified pyruvate dehydrogenase complex, which affects the tricarboxylic acid cycle, triggers proteotoxic stress, and induces cell death. Cellular cuprous can catalyze the substrate to produce fluorescent substances, and the higher the copper ion concentration, the more fluorescent substances are generated per unit time. The fluorescence delete can be detected at the excitation wavelength of 395 nm and emission wavelength of 480 nm.
The senescence-associated β-galactosidase (SA-β-gal) is a key biomarker of cellular senescence. Studies have shown that the activity of this enzyme significantly increases in senescent cells and is related to abnormal lysosomal function. It is commonly used to detect the state of cellular senescence and study the mechanism of aging. This kit can detect the activity of SA-β-gal in cells through a simple fluorescence method. The probe can enter the cell through the cell membrane, and SA-β-gal hydrolyzes the probe to emit fluorescence. The fluorescence intensity can be detected using a flow cytometer to determine the activity of SA-β-gal.
Cellulase (CL) decomposed the substrate to produce raw sugar. Under the action of the enzyme, it formed colored substances with the chromogen. The activity of CL was measured by the absorbance at 505 nm.
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