Bioenergetics Assays

Create a full mitochondrial function and toxicity profile with Cayman's simple, cost effective package of assay kits developed to screen potential inhibitors of the electron transport chain, to assess mitochondrial function in cells by detecting the rate of oxygen consumption, and to measure mitochondrial membrane potential. Cayman can also create a mitochondrial function and toxicity profile for you through our Bioenergetics Screening service. For more information, read our application note, Screening for Drug Induced Mitochondrial Dysfunction.

Oxygen Consumption Rate Assays In partnership with Luxcel Logo

  • Measure oxygen consumption rate in living cells
  • Novel phosphorescent oxygen probe, MitoXpress®-Xtra
  • Plate reader-based format precludes use of costly oxygen electrodes
  • Ideal for high throughput screening of mitochondrial uncouplers and inhibitors
MitoCheck® Mitochondrial OCR Assay Kit Product Image

MitoCheck® Mitochondrial OCR Assay Kit


Item №: 701170

Cayman’s Mitocheck® Mitochondrial OCR Assay Kit is designed to measure OCR directly in freshly isolated mitochondria. This kit combines a mitochondrial respiration buffer, substrates, and inhibitors with the phosphorescent oxygen probe MitoXpress® Xtra to accurately determine mitochondrial OCR in a high throughput manner. While there are several well-established methods that allow for the measurement of cellular OCR in cells, these methods only accurately reflect the respiration rates of the cell and may not necessarily represent specific mitochondrial function. For convenience, a mitochondrial tissue isolation procedure is outlined in this kit. However, the buffers needed to perform the isolation, which are available in Cayman’s Mitocheck® (Tissue) Mitochondrial Isolation Kit (Item No. 701010), must be purchased separately.

Oxygen Consumption Rate Assay Kit (MitoXpress® Xtra HS Method) Product Image

Oxygen Consumption Rate Assay Kit (MitoXpress® Xtra HS Method)


Item №: 600800

The oxygen consumption rate (OCR) of cells is an important indicator of normal cellular function. It is used as a parameter to study mitochondrial function as well as a marker of factors triggering the switch from healthy oxidative phosphorylation to aerobic glycolysis in cancer cells. Oxygen consumption is traditionally measured by a cumbersome oxygen electrode, a specialized piece of equipment that typically yields low sample throughput. The phosphorescent oxygen probe, MitoXpress® Xtra, developed by Luxcel Biosciences offers a novel method for analyzing oxygen consumption in whole cells. Cayman’s cell-based Oxygen Consumption Rate Assay Kit (MitoXpress® Xtra HS Method) utilizes this newly developed phosphorescent oxygen probe to measure oxygen consumption rate in living cells. Antimycin A, an inhibitor of the mitochondrial electron transport chain, is included to be used as a positive control. Glucose oxidase is also included in the kit to be used as a reference for oxygen depletion. The kit is easy to use and can be easily adapted to high throughput screening for compounds which modulate oxygen consumption rate.

Oxygen Consumption/MitoMembrane Potential Dual Assay Kit Product Image

Oxygen Consumption/MitoMembrane Potential Dual Assay Kit


Item №: 600880

Cayman’s Oxygen Consumption/Mitochondrial Membrane Potential Dual Assay Kit is a multiplex assay designed to provide a comprehensive picture of mitochondrial performance in living cells. This assay simultaneously measures both oxygen consumption rate and mitochondrial membrane potential by utilizing MitoXpress® Xtra, a phosphorescent oxygen probe, and the cationic dye, JC-1. Antimycin A, an inhibitor of the mitochondrial electron transport chain, is included to be used as a positive control. Additionally, glucose oxidase is provided as a reference for oxygen depletion. This easy-to-use kit can be adapted for high throughput screening of compounds that modulate mitochondrial function.

Oxygen Consumption/Glycolysis Dual Assay Kit Product Image

Oxygen Consumption/Glycolysis Dual Assay Kit


Item №: 601060

Cayman’s Oxygen Consumption/Glycolysis Dual Assay Kit is a multi-parameter approach to measure cellular oxygen consumption and glycolysis in living cells. This assay utilizes MitoXpress® - Xtra, a phosphorescent oxygen probe, to measure oxygen consumption rate, and also quantifies extracellular lactate as a readout for glycolysis. Antimycin A, an inhibitor of the mitochondrial electron transport chain, is included as a control. The kit can be used for efficient screening of compounds that modulate mitochondrial and glycolytic function in cultured cells.

MitoCheck® Mito Stress Test (ACU format) Product Image

MitoCheck® Mito Stress Test (ACU format)


Item №: 601450

Cayman's MitoCheck® Mito Stess Test (ACU format) is designed to measure oxygen consumption rate (OCR) and extracellular acidification (ECA) using MitoXpress® Xtra and pH-Xtra, respectively. OCR and ECA measurements occur simultaneously, and in real time under tightly controlled atmospheric conditions using open-flow respirometry.1,2,3,4 Included in this kit are the control compounds oligomycin (Complex V inhibitor), FCCP (uncoupler, increases OCR), and antimycin A (inhibits OCR) for easy assessment of mitochondrial health. This assay has been designed to work exclusively with the BMG LABTECH CLARIOstar® microplate reader equipped with dual injection ports, TRF detection, and ACU.

Glucose Metabolism Assays

  • Simple, reproducible, and sensitive tools for assaying glucose metabolism
  • Study modulators of cellular glucose uptake
Glucose Colorimetric Assay Kit Product Image

Glucose Colorimetric Assay Kit


Item №: 10009582

Glucose, a monosaccharide (or simple sugar), is the most important carbohydrate in biology. Transported via the blood stream, it is the primary source of energy for the body’s cells. Glucose level is tightly regulated in the human body. Failure to maintain blood glucose in the normal range leads to conditions of persistently high (hyperglycemia) or low (hypoglycemia) blood sugar. Diabetes mellitus, characterized by persistent hyperglycemia, is the most prominent disease related to failure of blood sugar regulation. Cayman’s Glucose Colorimetric Assay Kit provides a simple, reproducible, and sensitive tool for assaying glucose in plasma, serum, and urine. The glucose assay uses the glucose oxidase-peroxide reaction for the determination of glucose concentrations. In this assay, glucose is oxidized to δ-gluconolactone with concomitant reduction of the flavin adenine dinucleotide (FAD)-dependent enzyme glucose oxidase. The reduced form of glucose oxidase is regenerated to its oxidized form by molecular oxygen to produce hydrogen peroxide. Finally, with horseradish peroxidase as a catalyst, hydrogen peroxide reacts with 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to generate a pink dye with an optimal absorption at 514 nm.

Glucose Uptake Cell-Based Assay Kit Product Image

Glucose Uptake Cell-Based Assay Kit


Item №: 600470

Glucose metabolism is a primary source of energy and biomaterials for the maintenance of cell homeostasis. Extra glucose is stored in the muscles and liver as glycogen which is hydrolyzed to glucose and released into the blood when needed. The rate of glucose uptake in cells is dynamic and tightly regulated by hormones and/or growth factors including insulin. Cancer cells exhibit increased glucose uptake and metabolism by aerobic glycolysis in order to support a high rate of proliferation. Chemicals that block glucose uptake by cancer cells have been shown to have anti-cancer effects. Cayman’s Glucose Uptake Cell-based Assay Kit provides a convenient tool for studying modulators of cellular glucose uptake. The kit employs 2-NBDG, a fluorescently-labeled deoxyglucose analog, as a probe for the detection of glucose taken up by cultured cells. Apigenin, a flavonoid that has been reported to be an inhibitor of glucose transport, is included as a control.

Glucose-6-Phosphate Dehydrogenase Activity Assay Kit Product Image

Glucose-6-Phosphate Dehydrogenase Activity Assay Kit


Item №: 700300

Glucose-6-phosphate dehydrogenase (G6PDH) is a cytosolic enzyme that catalyzes the first step in the pentose phosphate pathway. This pathway includes converting glucose to ribose-5-phosphate, a precursor to RNA, DNA, ATP, CoA, NAD, and FAD. The pathway also generates NADPH. G6PDH deficiency, the most common enzyme deficiency worldwide, causes a spectrum of diseases including neonatal hyperbilirubinemia, acute hemolysis, and chronic hemolysis. G6PDH activity has been shown to be upregulated in rat and mouse models of obesity, hyperglycemia, and hyperinsulinemia. Cayman’s Glucose-6-Phosphate Dehydrogenase Assay provides a fluorescence-based method for detecting G6PDH activity in a variety of samples including erythrocyte lysates, tissue homogenates, and cell culture samples.

Glucose-6-Phosphate Fluorometric Assay Kit Product Image

Glucose-6-Phosphate Fluorometric Assay Kit


Item №: 700750

Glucose-6-phosphate (G6P) plays a key role in the homeostatic regulation of blood glucose levels by participating in glycolysis, glycogen synthesis, gluconeogenesis, and can be metabolized to NADPH, enabling protection against oxidative damage. Disruption of G6P activity leads to glycogen storage disease type I or von Gierke’s disease, a group of inherited metabolic diseases characterized by severe hypoglycemia, growth retardation, and hepatomegaly, due to accumulation of glycogen and fat in the liver. Cayman’s Glucose-6-Phosphate Fluorometric Assay provides a fluorescence-based method for detecting G6P in tissue homogenates and cell culture samples. In the assay, G6PDH catalyzes the oxidation of G6P to 6-phospho-D-gluconate, along with the concomitant reduction of NADP+ to NADPH. NADPH reacts with the fluorometric detector to yield a highly fluorescent product which can be analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm.

pH-XtraTM Glycolysis Cell-Based Assay Kit Product Image

pH-XtraTM Glycolysis Cell-Based Assay Kit Exclusive


Item №: 601230

Cayman’s pH-Xtra™ Glycolysis Cell-Based Assay Kit is designed to measure the extracellular acidification rate (ECAR), which is proportional to the rate of lactate production.1 This kit combines a glucose-based respiration buffer and inhibitors along with the fluorescent pH probe pH-Xtra™ allowing for the accurate determination of glycolytic rate in a high-throughput manner.2

Mitochondrial Membrane Potential Assays

  • A straightforward test to measure mitochondrial health in whole cells
  • Monitor the shift in the mitochondria-specific dye, JC-1, from green to red fluorescence as membrane potential increases
  • Quick and efficient, staining is usually achieved within 15 minutes
  • Applicable to flow cytometry, fluorescence microscopy, and 96-well plate formats
JC-1 Mitochondrial Membrane Potential Assay Kit Product Image

JC-1 Mitochondrial Membrane Potential Assay Kit


Item №: 10009172

Mitochondrial membrane potential, (ΔψM), is an important parameter of mitochondrial function used as an indicator of cell health. JC-1 is a lipophilic, cationic dye that can selectively enter into mitochondria and reversibly change color from green to red as the membrane potential increases. In healthy cells with high mitochondrial (ΔψM), JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the other hand, in apoptotic or unhealthy cells with low (ΔψM), JC-1 remains in the monomeric form, which shows only green fluorescence. Cayman’s JC-1 Mitochondrial Membrane Potential Assay Kit provides all the necessary reagents, as well as complete instructions, for analysis of mitochondrial integrity in whole cells. The assay is applicable to fluorescence microscopy and fluorescent plate reader. For flow cytometry, Cayman’s JC-1 Mitochondrial Membrane Potential Flow Cytometry Assay Kit (Item No. 701560) is recommended.

JC-1 Mitochondrial Membrane Potential Flow Cytometry Assay Kit Product Image

JC-1 Mitochondrial Membrane Potential Flow Cytometry Assay Kit New


Item №: 701560

Cayman’s JC-1 Mitochondrial Membrane Potential Flow Cytometry Assay Kit can be used to study mitochondrial behavior in a variety of conditions, including apoptosis. Changes in ΔψM, reflected by aggregation level of JC-1, can be determined as a ratio of red to green mean fluorescence intensities using flow cytometry. Flow cytometry is an ideal way to assess the JC-1 aggregation at the single-cell level, and provides the added benefit of potential to multiplex other readouts from the same cells.3 This kit additionally contains FCCP for treatment of cells as a compensation control. The reagents in this kit are sufficient for staining up to 500 samples for JC-1.

ETC Activity AssaysMitoCheck Logo

  • Screen your compound for potential inhibition of the electron transport chain
  • Designed as a colorimetric 96-well plate format, no special equipment required
  • Isolated bovine heart mitochondria are provided in the kit
  • Preincubation with antibodies is not required
  • Amenable to high throughput screening
MitoCheck® Complex I Activity Assay Kit Product Image

MitoCheck® Complex I Activity Assay Kit


Item №: 700930

Complex I (NADH oxidase/Co-enzyme Q reductase) is one of the major sites of electron entry into the mitochondrial electron transport chain (ETC). Complex I catalyzes the 2 electron oxidation of NADH followed by the reduction of ubiquinone (Q) to form ubiquinol (QH2), and ultimately the reduction of the terminal electron acceptor, O2. During the passage of electrons from NADH to Q, the translocation of four protons (H+) from the mitochondrial matrix to the intermembrane space occurs, contributing to the chemiosmotic proton gradient, which is required for oxidative phosphorylation. Cayman's MitoCheck® Complex I Activity Assay allows for the activity of complex I to be determined without the need to isolate mitochondria or pre-incubate with antibodies. The rate of NADH oxidation is measured by a decrease in absorbance at 340 nm and is proportional to the activity of complex I. For the use of this kit with other types of tissue mitochondria, please see Cayman's MitoCheck® Mitochondrial (Tissue) Isolation Kit (Item No. 701010).

MitoCheck® Complex II Activity Assay Kit Product Image

MitoCheck® Complex II Activity Assay Kit


Item №: 700940

Complex II (succinate dehydrogenase/co-enzyme Q reductase) is one of the major sites of electron entry into the mitochondrial electron transport chain (ETC). Complex II catalyzes the oxidation of succinate to fumarate, and in the process reduces ubiquinone (Q) to ubiquinol (QH2). Ultimately, oxidation of succinate will lead to reduction of O2, the terminal step in mitochondrial respiration. Cayman’s MitoCheck® Complex II Activity Assay allows for the activity of complex II to be determined without the need to isolate mitochondria or pre-incubate with antibodies. As complex II oxidizes succinate, electrons are passed to an analog of ubiquinone and then on to DCPIP, which, when oxidized, absorbs in the 600 nm range. The absorbance of DCPIP will decrease upon reduction. Thus, Complex II activity is measured as a decrease in absorbance at 600 nm over time. For the use of this kit with other types of tissue mitochondria, please see Cayman's MitoCheck® Mitochondrial (Tissue) Isolation Kit (Item No. 701010).

MitoCheck® Complex II/III Activity Assay Kit Product Image

MitoCheck® Complex II/III Activity Assay Kit


Item №: 700950

Complex III (CoQ cytochrome c oxidoreductase) is an essential protein for mitochondrial oxidative phosphorylation. Complex III functions as both a gatekeeper for mitochondrial respiration and as a major source of reactive oxygen species III. It accepts electrons from complexes I and II in the form of QH2, the reduced form of the electron carrier ubiquinone. Once bound to complex III, QH2 undergoes a series of redox reactions, known as the Q-cycle. During the Q-cycle, electrons are passed from QH2 onto cytochrome c via the Rieske iron-sulfur protein and cytochrome c1 resulting in the translocation of 4H+ and the generation of 02•-. Cayman's MitoCheck® Complex II/III Activity Assay measures the reduction of excess cytochrome c (550 nm absorbance) as catalyzed by complex III. This assay is coupled to succinate co-enzyme Q oxidoreductase (complex II) for the generation of QH2. Due to the dependence on complex II activity, a counter-screen for complex II activity (Item No. 700940) should be performed in order to truly measure complex III activity. For the use of this kit with other types of tissue mitochondria, please see Cayman's MitoCheck® Mitochondrial (Tissue) Isolation Kit (Item No. 701010).

MitoCheck® Complex IV Activity Assay Kit Product Image

MitoCheck® Complex IV Activity Assay Kit


Item №: 700990

Complex IV (cytochrome c oxidase) is the terminal electron acceptor in the mitochondrial electron transport chain. Complex IV functions by oxidizing cytochrome c and completing a four-electron reduction of oxygen (O2) to form water. During this process, two H+ are translocated from the mitochondrial matrix to the intermembrane space, contributing the mitochondrial membrane potential required for ATP synthesis. Common inhibitors of complex IV include potassium cyanide, azide, O2 limitation, nitric oxide, and carbon monoxide. Cayman’s MitoCheck® Complex IV Activity Assay is designed to measure the direct oxidation of cytochrome c by complex IV, in an isolated bovine heart mitochondrial system that is supplied within the kit. For the use of this kit with other types of tissue mitochondria, please see Cayman's MitoCheck® Mitochondrial (Tissue) Isolation Kit (Item No. 701010).

Mitochondrial IsolationMitoCheck
                Logo

  • Isolate intact, functional mitochondria from heart, liver, kidney, or muscle tissue
  • Complete package of reagents and step-by-step instructions
  • Isolated coupled mitochondria are suitable for use in:
    • Oxygen consumption experiments
    • All Cayman MitoCheck Activity Assays
MitoCheck® Mitochondrial (Tissue) Isolation Kit Product Image

MitoCheck® Mitochondrial (Tissue) Isolation Kit


Item №: 701010

When measuring mitochondrial function or the effects of an unknown on mitochondrial function, it can be difficult to determine a mechanism using whole organisms or tissue. When this is the case, isolated mitochondria provide a simple and biochemically relevant experimental model. Cayman’s MitoCheck® Mitochondrial (Tissue) Isolation Kit includes a complete package of reagents and step-by-step instructions for isolating coupled mitochondria* from freshly harvested mammalian heart, liver, or kidney tissue through a process of differential centrifugation in isotonic buffers. *Coupled mitochondria are defined as mitochondria capable of phosphorylating ADP at the F1FO-ATP synthase (Complex V) through the utilization of a proton (H+) gradient, which is generated by the electron transport chain (ETC) in the presence of appropriate substrates (e.g., succinate) and in the absence of uncouplers and mitochondrial inhibitors.

Citrate Synthase ActivityMitoCheck
                Logo

  • Simple and convenient determination of citrate synthase activity from isolated mitochondria or cell homogenates
  • Measures the production of SH-CoA by monitoring the absorbance of Citrate Synthase Developing Reagent at 412 nm in a convenient 96-well format
MitoCheck® Citrate Synthase Activity Assay Kit Product Image

MitoCheck® Citrate Synthase Activity Assay Kit


Item №: 701040

The condensation of the dicarboxylate oxaloacetate and acetyl CoA to the tricarboxylate citrate is catalyzed by citrate synthase. It is within this reaction that carbon molecules (as acetyl CoA) obtained from pyruvate oxidation are fed into the tricarboxylic acid (TCA or citric acid) cycle. As a mitochondrial enzyme, citrate synthase is commonly used as a normalization factor for mitochondrial protein, but can also be used as a biomarker for mitochondrial content in a tissue homogenate. Cayman's MitoCheck® Citrate Synthase Activity Assay Kit allows for the simple and convenient determination of citrate synthase activity from isolated mitochondria, tissue, or cell homogenates. This assay measures the production of SH-CoA by monitoring the absorbance of Citrate Synthase Developing Reagent at 412 nm in a convenient 96-well format.

Complex V Activity AssayMitoCheck
                Logo

  • Measure the activity of the mitochondrial Complex V (F1FoATP synthase)
  • Isolated bovine heart mitochondria are provided
  • Tissue mitochondria can also be acquired for use in this assay by using Cayman's MitoCheck® Mitochondrial (Tissue) Isolation Kit
MitoCheck® Complex V Activity Assay Kit Product Image

MitoCheck® Complex V Activity Assay Kit


Item №: 701000

The mitochondrial F1FO ATP synthase, commonly referred to as complex V, is a multi-subunit protein that utilizes energy in the form of a proton gradient generated by the electron transport chain, to phosphorylate ADP to form ATP in the presence of Pi. This mechanism, referred to as oxidative phosphorylation, is responsible for ~90% of ATP generation in mammalian cells. Cayman's MitoCheck® Complex V Activity Assay measures the activity of Complex V, in the reverse reaction, as an ATPase in the mitochondrial samples provided. In this assay, ATP is dephosphorylated ADP by complex V. The ADP is then utilized by pyruvate kinase to convert phosphoenolpyruvate into pyruvate with the concomitant generation of ATP. Pyruvate, in the presence of NADH and lactate dehydrogenase, is then reduced to lactate and NAD+. The rate of NADH oxidation is monitored at 340 nm. Isolated bovine heart mitochondria are provided in this kit. Tissue mitochondria can also be acquired using Cayman’s MitoCheck® Mitochondrial (Tissue) Isolation Kit (Item No. 701010).

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