Cell Mito Stress Test
Cell Mito Stress Test - Related Videos
10:39
Purification of Mitochondria from Yeast Cells
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Cited by 85 •
2009
We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.
13:42
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
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Cited by 19 •
2012
A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.
11:13
MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells
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Cited by 47 •
2017
Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.
09:34
Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells
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Cited by 59 •
2016
Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.
05:37
Visualization of Mitochondria within Intraepidermal Nerve Fibers
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2025
Source: Hamid, H. S., et al. Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers. J. Vis. Exp. (2017) This video demonstrates the method for visualizing nerve-specific mitochondria using three-dimensional imaging. It outlines the process of acquiring sequential scans of pre-labeled intraepidermal nerve fibers and generating 3D surface images to isolate and display mitochondria in relation to nerve structures.
FAQs
Each compound reveals a different component of oxygen consumption. Oligomycin inhibits ATP synthase, so the resulting change distinguishes respiration linked to ATP production from proton leak. FCCP uncouples respiration and exposes maximal respiratory capacity. Rotenone plus antimycin A blocks the electron transport chain, allowing non-mitochondrial respiration to be identified and separated from mitochondrial oxygen consumption.
Spare respiratory capacity is derived by comparing maximal respiration after FCCP with the cell’s basal respiration. It indicates how much additional respiratory output remains available when energy demand increases. In immune or infected-cell experiments, differences in this parameter can help characterize whether metabolic reprogramming changes the cell’s ability to respond to increased energetic demands.
The assay assigns oxygen consumption to functional categories by comparing measurements across the inhibitor sequence. Oligomycin-related changes estimate respiration associated with ATP synthesis and help identify proton leak, while rotenone and antimycin A establish the non-mitochondrial component. Together with basal and FCCP-stimulated values, these comparisons provide a profile rather than a single respiration measurement.
Sequential addition creates a controlled series of respiratory states in the same living-cell measurement. Basal respiration is recorded before mitochondrial modulation, ATP synthase inhibition tests the contribution of ATP production, uncoupling challenges the system, and electron transport chain blockade defines the residual signal. This order supports calculation of multiple related metabolic parameters from one assay.
The workflow uses living cells and extracellular flux analysis to record oxygen consumption while mitochondrial modulators are introduced sequentially. Measurements begin under basal conditions, followed by oligomycin, FCCP, and then rotenone with antimycin A. The resulting oxygen-consumption profile is used to calculate basal and maximal respiration, ATP-linked respiration, proton leak, spare capacity, and non-mitochondrial respiration.
Researchers can compare respiration profiles in immune cells exposed to pathogens or inflammatory stimuli, or examine how infection changes host-cell metabolism. Shifts in basal respiration, maximal capacity, ATP-linked respiration, proton leak, or spare capacity provide functional evidence of metabolic reprogramming. These measurements help connect cellular energy production with host responses and immune-cell activity.