Study Overview
We have introduced the extracellular flux analyzer XFe96 (Agilent Technologies), which allows us to evaluate the energy metabolic state of cells in real time.
1. Mitochondrial respiration assessment
The XF Stress Kit measures key indicators of mitochondrial function: basal respiration, ATP production, proton leak, and maximal respiration (spare respiratory capacity).
2. Glycolysis Assessment
The XF Glycolytic Rate Kit can be used to measure glycolytic activity from the rate of glycolysis-derived proton flux. Basal glycolytic capacity and reserve glycolytic capacity can be measured.
3. ATP production assessment
The XF Real-Time ATP Rate Kit can be used to measure not only the cellular ATP production rate, but also the proportion of ATP produced by mitochondrial respiration and glycolysis. It can measure mitoATP production rate, glycoATP production rate, and total ATP production rate.
4. Assessment of utilization of major respiratory substrates
The XF Mito Fuel Flex Kit can be used to analyze the utilization of the major respiratory substrates: glucose, fatty acids, and glutamine.
Three main indices can be measured:
Fuel Dependency (the utilization rate of a specific substrate under basal conditions)
Fuel Flexibility (the difference between Capacity and Dependency)
Fuel Capacity (the ratio of specific substrate utilization to energy demand)
Explanation of terms
1. What is ATP?
Adenosine triphosphate, which plays an important role in the release and storage of energy in living organisms, as well as in the metabolism and synthesis of substances.
2. What is mitochondrial respiration?
Electrons such as NADH2+ produced in the TCA cycle are transported between oxygen on the inner mitochondrial membrane (electron transport chain) to produce large amounts of ATP. Oxygen is consumed in this process.
3. What is glycolysis?
ATP is produced during the process of breaking down glucose taken into cells into pyruvate in the cytoplasm. This is followed by the excretion of lactate outside the cell. During this pathway, hydrogen ions from the lactate excreted outside the cell cause the extracellular pH to become acidic.
Equipment used
XFe96 (Agilent Technologies)
Features of the extracellular flux analyzer
■ Real-time measurement of metabolism in living cells
■ Simultaneous evaluation of mitochondrial respiration (OXPHOS) and glycolysis
■ Functional metabolic evaluation using chemical compounds
■ High-throughput
Typical analysis examples
・Cancer research
Evaluating metabolic changes characteristic of cancer cells, etc.
・Immunology Research
Evaluating pro-inflammatory and anti-inflammatory macrophages
Evaluating T cell activation, etc.
・Obesity Research
Evaluating the differentiation and function of brown and beige adipocytes
Evaluating the energy substrates used by cells, etc.
・Mitochondrial Function Assessment
Evaluation of mitochondrial function by measuring basal and maximal mitochondrial respiration, etc.
References
●Selective Cancer Therapy Effects of Vitamin C
Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH
Yun et al., Science. 2015
●CAR-T Cancer Treatment
Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy
Wei et al., Nature. 2019
●iPSC Survival, Differentiation, and Substrate Dependence (Selectivity)
Glutamine Oxidation is indespensable for Survival of Human Pluripotent Stem Cells
Tohyama et al., Cell Metabolism. 2016
*Studies will be mainly conducted by those with doctoral degrees.
