Mitochondria Research DOJINDO LABORATORIES

Science NoteMitochondrial and Metabolic Activity Indicators

Molecular Control of Mitochondrial Ca²⁺ Efflux in Neurons [Mar. 18, 2026]

Mitochondrial Ca²⁺ control in neurons links activity to metabolism and is important for neuronal function and memory. Defining the proteins that regulate mitochondrial Ca²⁺ flux is therefore important for understanding cognitive decline and neurodegeneration. Recent studies showed that loss of the mitochondrial H⁺/Ca²⁺ exchanger Letm1 reduces Ca²⁺ efflux, thereby sustaining mitochondrial Ca²⁺ signals, extending metabolic activation, and enhancing long term memory in flies and mice. Another study showed that TMEM65 is necessary and sufficient for mitochondrial Na⁺/Ca²⁺ exchange, thereby identifying the molecular basis of mito-NCX, a major neuronal mitochondrial Ca²⁺ efflux pathway. Together, these findings clarify major regulators of neuronal mitochondrial Ca²⁺ efflux relevant to dementia related biology.

Mitochondrial Ca²⁺ efflux controls neuronal metabolism and long-term memory across species (Nature Metabolism, 2026)
Summary: Neuronal mitochondrial Ca²⁺ normally acts as a transient metabolic signal during activity, but whether its clearance influences long-term memory remains unclear. This study shows that Letm1, a mitochondrial H⁺/Ca²⁺ exchanger involved in Ca²⁺ efflux, prolongs metabolic activation when depleted, promotes long-term memory formation in flies and mice, and suggests relevance to memory decline in neurodegenerative disorders.

Highlighted technique: Pyruvate enters mitochondrial metabolism through the TCA cycle, so the authors measured pyruvate flux to test whether Letm1 depletion truly increases mitochondrial metabolism, not just ATP levels. They expressed Pyronic, a FRET-based pyruvate sensor, in rat hippocampal neurons, blocked mitochondrial metabolism with sodium azide, and used the rate of cytosolic pyruvate buildup as a readout of pyruvate flow into mitochondria.

TMEM65 functions as the mitochondrial Na⁺/Ca²⁺ exchanger (Nature Cell Biology, 2025)
Summary: Mitochondrial Na⁺/Ca²⁺ exchange (mito-NCX) is the main pathway for clearing mitochondrial Ca²⁺ in neurons, but its molecular identity has remained unresolved despite NCLX being widely viewed as the leading candidate. This study shows that TMEM65 is necessary and sufficient for mito-NCX, thereby strongly identifying TMEM65 as the exchanger itself and redefining the molecular basis of mitochondrial Ca²⁺ efflux relevant to neurodegeneration and dementia.

Highlighted technique: TThe authors evaluated mitochondrial NCX (mito-NCX) activity by treating HEK293 cells with altered TMEM65 expression with digitonin, which permeabilized the plasma membrane while preserving mitochondria, and then monitoring Na+-induced Ca²⁺ release into the external medium using Calcium Green-5N. They also isolated mitochondria from the cells and measured total mitochondrial Ca²⁺ by extracting Ca²⁺ with BAPTA under denaturing conditions in the presence of SDS.

Mitochondrial and Metabolic Activity Indicators (click to open/close)

Target	Kit & Probes
Mitochondrial and Metabolic Activity Indicators	Calcium Kit II - Fluo 4 and Calcium Kit II - Fura 2
Glycolysis/Oxidative phosphorylation Assay	Glycolysis/OXPHOS Assay Kit
Oxygen consumption rate assay	Extracellular OCR Plate Assay Kit
Intracellular ATP Assay	ATP Assay Kit-Luminescence
Intact Mitochondria Fractionation	IntactMito Fractionation Kit for Tissue
MitoComplex-I Activity Assay	MitoComplex-I Activity Assay Kit
Mitochondrial Staining	MitoBright LT Green / Red / Deep Red
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection

Application Note I (click to open/close)
> Inhibition of Mitochondrial Electron Transport Chain

Antimycin stimulation of Jurkat cells was used to evaluate the changes in cellular state upon inhibition of the mitochondrial electron transport chain using a variety of indicators.

The results showed that inhibition of the electron transport chain resulted in (1) a decrease in mitochondrial membrane potential and (2) a decrease in OCR. In addition, (3) the NAD⁺/NADH ratio of the entire glycolytic pathway decreased due to increased metabolism of pyruvate to lactate to maintain the glycolytic pathway, (4) GSH depletion due to increased reactive oxygen species (ROS), and (6) increase in the NADP⁺/NADPH ratio due to decreased NADH required for glutathione biosynthesis were observed.

Application Note II (click to open/close)
> Activity Evaluation of Mitochondria Fractionated from Mouse Brain

Mitochondria were isolated from mouse brain tissue, and oxygen consumption rate (OCR), mitochondrial membrane potential (MMP), and Complex I activity were measured.

The results showed that the addition of succinate, a substrate that activates Complex II of the electron transport chain, increased both OCR and MMP. In contrast, FCCP treatment reduced MMP, indicating that intact mitochondria were successfully fractionated.
Furthermore, in the Complex I activity assay, a decrease in activity was observed following treatment with rotenone, a Complex I inhibitor.

＜Product used＞
Mitochondrial Fractionation: IntactMito Fractionation Kit for Tissue (Code: MT17)
　OCR measurement: Extracellular OCR Plate Assay Kit (Code: E297)
　MMP detection: JC-1 MitoMP Detection Kit (Code: MT09)
　Complex I activity assay: MitoComplex- I Activity Assay Kit (Code: MT18)

＜Experimental Conditions＞
OCR Measurement
　 Amount of mitochondria: 50 μg/well (as protein levels)
　 Succinate: 10 mmol/l
MMP Detection
　 Amount of mitochondria: 50 μg/well (as protein levels)
　 Succinate: 10 mmol/l,   FCCP: 4 μmol/l
Complex I Activity Assay
　 Amount of mitochondria: 20 μg/well (as protein levels)
Rotenone: 10 μmol/l

Topics

Why is Mitochondrial Research Important?
Selection Guide for Mitochondria-related Reagents
Cancer, Cell Death, and Mitochondria
Mitochondrial Function and Cellular Senescence
Neurodegeneration-Autophagy and Mitochondrial Damage

Why is Mitochondrial Research Important?

Mitochondrial analysis is essential for understanding cellular energy metabolism because mitochondria regulate ATP production, redox balance, and signaling pathways. Thus, assessment of mitochondrial functions such as membrane potential, mitophagy, and respiration provides insight into cellular abnormalities, and these studies are critical in neurodegenerative diseases, cancer biology, and metabolic disorders, helping to uncover therapeutic targets and disease mechanisms.

Selection Guide for Mitochondria-related Reagents

Mitochondria research is very multi-faceted, because the multi-functional organelle is not only involved in energy production in a cell, but other additional cellular functions. The active cycle of mitochondrial fusion and division induces morphological changes, which is called mitochondrial dynamics. Abnormalities in morphological control of mitochondria are associated with neurodegenerative diseases, metabolic disorders, aging, and so on. Therefore, the demand for long-term observation of mitochondrial dynamics has recently been increasing.

Selection Guide of Reagents

The following table lists reagents for mitochondrial research designed to stain and detect mitochondria (MitoBright LT, MitoTracker, etc.), mitochondrial membrane potential (JC-1, TMRM, TMRE, etc.), reactive oxygen species AKA ‘ROS’ (MitoBright ROS, MitoSOX, etc.), mitophagy, and lipid peroxides.

Mitophagy

Mitophagy
Reagent	Mtphagy Dye	Keima-Red
Principle	Mtphagy Dye (included in Mitophagy Detection Kit) is a pH-sensitive fluorescent probe that accumulates in mitochondria and emits red fluorescence due to acidic conditions in a lysosome.	A pH-sentitive ratiometric fluorescent protein. The excitation spectrum changes accoring to pH. This protein shows high fluorescence ratio (Ex. 550 nm/440 nm) values in a lysozome.
Fixed cell staining	–	–
Live-cell staining	Yes	Yes
Fixation after live-cell staining	–	–
Staining time	> 30 min	–
Ex / Em	530 / 700	440, 550 / 620
Product code	MD01, MT02	–

ROS

Lipophilic peroxide / Singlet oxygen / Superoxide

	Lipophilic peroxide	Singlet oxygen	Superoxide	Superoxide
Reagent	MitoPeDPP	Si-DMA	MitoBright ROS Deep Red	MitoSOX
Principle	A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with lipophilic peroxides in mitochondria to emit fluorescence.	A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with singlet oxigen generated in mitochondria to emit red fluorescence.	A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit fluorescence.	A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit red fluorescence.
Fixed cell staining	–	–	–	–
Live-cell staining	Yes	Yes	Yes	Yes
Fixation after live-cell staining	–	–	–	–
Staining time	> 15 min	> 45 min	> 10 min	> 10 min
Ex / Em	452 / 470	644 / 670	540 / 670	510 / 590
Product code	M466	MT05	MT16	–

Membrane potential

Membrane potential
Reagent	JC-1	MT-1	TMRM, TMRE
Principle	A cell-permeant ratiometric fluorescent dye that accumulates in intact mitochondria due to the membrane potential. The excitation spectrum changes according to the mitochondria membrane potential.	Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. MT-1 is extremely photostable and more sensitive than JC-1 and can provide equivalent detection sensitivity to TMRE.	Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Diffusion of the probes occurs in a damaged mitochondria that has the decreased membrane potential.
Fixed cell staining	–	–	–
Live-cell staining	Yes	Yes	Yes
Fixation after live-cell staining	–	Yes	–
Staining time	30-60 min	30 min	30-60 min
Ex / Em	Monomer: 514 / 529 J-aggregation: 585/590	530-560 / 570-640	550 / 575
Product code	MT09	MT13	–

Mitochondria staining

Mitochondria staining
Reagent	MitoBright LT series	MitoBright IM Red	MitoTracker series	Rhodamine 123
Principle	Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential.	Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential and covalently binds to proteins and other biomolecules.	Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential.	Cell-permeant fluorescent dye that accumulates in intact mitochondria due to the membrane potential.
Fixed cell staining	–	–	–	–
Live-cell staining	Yes	Yes	Yes	Yes
Fixation after live-cell staining	Yes	Yes	–	–
Staining time	30 min	30 min	15-45 min	> 15 min
Ex / Em	493 / 508, 547 / 563, 643 / 663	548 / 566	490 / 516 ~ 644 / 665	507 / 529
Product code	MT10, MT11,MT12	MT15	–	R233

Metal Ion Detection

	Iron ion (Fe²⁺)	Calcium ion (Ca²⁺)
Reagent	Mito-FerroGreen	Rhod2-AM
Principle	A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with ferrous ion in mitochondria to emit green fluorescence.	A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with calcium ion in mitochondria to emit red fluorescence.
Fixed cell staining	–	–
Live-cell staining	Yes	Yes
Fixation after live-cell staining	–	–
Staining time	30 min	30-60 min
Ex / Em	505 / 535	553 / 576
Product code	M489	R002

Reagents and Kits List

Application	Products
Mitophagy Detection	Mitophagy Detection Kit
Mitochondrial Phospholipid Peroxidase Detection	MitoPeDPP
Mitochondrial Ferrous Ion Detection	Mito-FerroGreen
Mitochondrial Superoxide	MitoBright ROS - Mitochondrial Superoxide Detection
Mitochondrial Singlet Oxygen Detection	Si-DMA for Mitochondrial Singlet Oxygen Imaging
Mitochondrial Membrane Potential	JC-1 MitoMP Detection Kit MT-1 MitoMP Detection Kit
Mitochondria Staining	MitoBright LT Green MitoBright LT Red MitoBright LT Deep Red MitoBright IM Red for Immunostaining

Cancer, Cell Death, and Mitochondria

Apoptosis is a protective defense mechanism that effectively suppresses tumor growth and eliminates tumor cells.
One of the main mechanisms that trigger apoptosis is the increase in mitochondrial metabolic activity, which leads to elevated ROS levels in cancer cells. Excessive ROS damage mitochondrial function, causing mitochondrial membrane depolarization, which subsequently activates the intrinsic apoptosis pathway. Tumor cell immune evasion is a key feature of tumor pathophysiology, and mitochondria play a central role in both inhibiting and promoting immune evasion within the complex mechanism s involved in immune responses.¹⁾

The Potential of Ferroptosis in Cancer Therapy: Many studies have found that ferroptosis sensitivity can be used to target tumors resistant to conventional therapies (such as triple-negative breast cancer and glioblastoma).²⁾

Ferroptosis and the Immune Microenvironment: Neutrophils in the tumour microenvironment die spontaneously by ferotosis and the lipid peroxide released suppresses T-cell activity, thereby suppressing tumour immunity.^{3)

Reference

1) Gao, J., Cancer Gene Therapy, 2024, 31, 970-983

2) Yang, F., Cell Metabolism, 2023, 35(1), 84-100

3) Kim, R., Nature, 2022, 612, 338-346}

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Mitochondrial Function and Cellular Senescence

In senescent cells, due to mitochondrial dysfunction, ATP is primarily generated through the anaerobic glycolysis pathway, leading to an increase in lactate production²⁾. DNA damage is one of the causes of mitochondrial dysfunction in cellular aging. The accumulation of DNA damage activates DNA repair mechanisms and increases NAD⁺ consumption. The decrease in NAD⁺ levels reduces SIRT1 activity, an important factor in maintaining mitochondrial function, leading to impaired mitochondrial function (inhibition of electron transfer → ATP production / reduction of NAD⁺ levels)^1),3).

Reference:

1. J. Wu, Z. Jin, H. Zheng and L. Yan, “Sources and implications of NADH/NAD+redox imbalance in diabetes and its complications”, Diabetes Metab. Syndr. Obes., 2016, 9, 145

2. Z. Feng, R. W. Hanson, N. A. Berger and A. Trubitsyn, “Reprogramming of energy metabolism as a driver of aging”, Oncotarget., 2016, 7(13), 15410.

3. S. Imai and L. Guarente, “NAD+ and sirtuins in aging and disease”, Trends in Cell Biology, 2014, 24(8), 464.

Neurodegeneration-Autophagy and Mitochondrial Damage

Indicator	Mitophagy	Mitochondrial fission abnormalities	ROS	MPTP (Mitochondrial Permeability Transition Pore)	MMP （Mitochondrial Membrane Potential）	ATP
Detection Kit	MD01 Mitophagy Detection Kit	MT10/MT11/MT12 MitoBright LT Green/Red/Deep Red	R252/R253 ROS Assay Kit	-	MT09:JC-1 MT13:MT-1	A550 ATP Assay Kit-Luminescence
Alzheimer's disease (AD)	↓	✓	↑	↑	↓	↓
Parkinson's disease (PD)	↓			-	-
Huntington's disease (HD)	-			-	-
Amyotrophic lateral sclerosis (ALS)	-			-	↓

Refernce :

Role of Mitochondria in NeurodegenerativeDiseases: From an Epigenetic Perspective

Mitophagy / Autophagy Analysis Products

Product Name	Probe 1 Dyes and Fluorescence Properties	Probe 2 Dyes and Fluorescence Properties
Mitophagy Detection Kit	Mtphagy Dye Ex: 500-560 nm, Em: 670-730 nm	Lyso Dye Ex: 350-450 nm, Em: 500-560 nm
Mtphagy Dye	Mtphagy Dye Ex: 500-560 nm / Em: 690-750 nm
Autophagic Flux Assay Kit*	DAPRed Ex: 500-560 nm / Em: 690-750 nm	DALGreen Ex: 350-450 nm / Em: 500-560 nm

^{*Autophagic Flux Assay Kit includes lysosome acidification Inhibitor as a negative control.}

Mitochondria Research