Multiplex your mitochondrial data

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Mitochondria can make up as much as 10% of the cell volume of eukaryotic cells. Changes in mitochondrial morphology and function are good indicators of cell health, and multiplexing mitochondrial morphology reagents with probes that assess function can provide more in-depth information about mitochondrial health. We have developed a wide range of Molecular Probes™ reagents to investigate mitochondria in both live- and fixed-cell imaging applications. In this article, we highlight a few of our most referenced mitochondrial probes.

Mitochondrial morphology probes

Mitochondria can have a fragmented morphology, with many spheroid- shaped mitochondria, or a reticulated morphology, in which the mitochondrion is a single, many-branched structure [1,2]. The number of mitochondria is a function of several variables, including cell type, cell cycle or differentiation stage, cellular energy level, and overall cell health. Simply staining mitochondria and observing their morphology through a microscope can provide a significant amount of information about their overall biology and functional state (Table 1). Figure 1 shows mitochondria with normal reticulated morphology, as well as spheroid-shaped mitochondria being cleared by autophagy.

Table 1. Various Molecular Probes tools to study mitochondrial morphology.

ProbeMechanism of action
MitoTracker™ Green, Red, and Deep Red probesMitoTracker Green (Cat. No. M7514), MitoTracker Red FM (Cat. No. M22425), and MitoTracker Deep Red FM (Cat. No. M22426) dyes are sequestered by functioning mitochondria. However, cells stained with these dyes retain their fluorescent staining patterns even if mitochondrial function is disrupted or if cells are subjected to fixation and permeabilization. This property makes them useful morphology markers that, once bound, are independent of mitochondrial function [3].
CellLight™ probesCellLight Mitochondria-GFP (Cat. No. C10600) and CellLight Mitochondria-RFP (Cat. No. C10601) stain mitochondria independent of their functional state. These probes use a one-step protocol—based on BacMam gene delivery and expression technology—to label all the mitochondria in cells with either GFP or RFP. After application, mitochondria can be imaged in live cells, or cells can be fixed and permeabilized for further study.
AntibodiesAntibodies are a powerful tool for staining mitochondria in fixed cells. Antibodies such as the anti–Complex V monoclonal antibody (Cat. No. 459000) can stain all mitochondria in cells for morphological studies.
 Figure 1. Mitochondrial morphology during mitophagy. HeLa cells labeled with Hoechst™ 33342 dye (blue) and expressing CellLight™ Mitochondria-GFP (green) and Premo™ Autophagy Sensor LC3B-RFP (red) were treated with CCCP to depolarize mitochondria. Loss of mitochondrial membrane potential triggers the targeted clearance of damaged mitochondria via mitophagy, as reflected through the colocalization of the autophagosomal marker LC3B-RFP with mitochondrial spheroids.

Mitochondrial functional tools

Mitochondrial dysfunction is associated with various diseases [4], and is a hallmark of cellular toxicity. We offer a variety of Molecular Probes reagents to study mitochondrial function from many perspectives, including probes for mitochondrial membrane potential, calcium flux, oxidative phosphorylation, autophagy/mitophagy, and cytosolic pH (Table 2).

Table 2. Various Molecular Probes tools to study mitochondrial function.

FunctionProbe recommendations
Mitochondrial membrane potentialTMRM (Cat. No. T668) is a classic dye for studying mitochondrial membrane potential because it accumulates in mitochondria with intact membrane potential and, upon loss of potential, leaks into the cytoplasm.
Mitochondrial calcium fluxThe calcium indicator rhod-2 AM (Cat. No. R1244) has long been used to measure mitochondrial calcium flux because of its preferential accumulation in mitochondria (Figure 3).
Oxidative phosphorylationMitochondria generate various reactive oxygen species (ROS), particularly superoxides [5]. MitoSOX™ Red dye (Cat. No. M36008) is a mitochondria-targeted superoxide sensor. CellROX™ Orange (Cat. No. C10443) and CellROX™ Deep Red (Cat. No. C10422) Reagents are general oxidative stress indicators, but their signals can be localized to mitochondria when used together with mitochondrial morphology probes (Figure 4).
Autophagy/mitophagyCells routinely recycle dysfunctional mitochondria through a specific autophagy process called mitophagy. This mechanism can be detected by multiplexing CellLight Mitochondria-GFP or CellLight Mitochondria-RFP with one of the Premo™ Autophagy Sensors (Figure 1).
Cytosolic pHDisruption of mitochondrial function can alter cytosolic pH [6]. pHrodo™ Green AM (Cat. No. P35373) or pHrodo™ Red AM (Cat. No. P35372) can be used to detect changes in cytosolic pH.

The power of multiplexing

The study of mitochondria can be improved by multiplexing functional probes with morphology probes. As an example, CellLight Mitochondria-GFP or CellLight Mitochondria-RFP can be combined with potential-sensitive dyes such as TMRM to monitor mitochondrial structural integrity while also assessing mitochondrial membrane potential (Figure 2). Figure 3 demonstrates the power of multiplexing the mitochondrial calcium flux indicator rhod-2 AM with potential-independent mitochondrial markers such as CellLight Mitochondria-GFP, which enables the visualization of mitochondrial fission, fusion, and motility before, during, and after calcium uptake. With the help of multiplexing, mitochondrial function can be determined even with tools that are not specifically targeted for mitochondria, when they are used in conjunction with mitochondria-targeted probes. When multiplexed with CellLight Mitochondria-GFP, CellROX Orange, a probe of general oxidative stress, can indicate the oxidative stress status of mitochondria (Figure 4).

Figure 2. Dynamic imaging of mitochondrial membrane potential and organelle integrity. HeLa cells were transduced with CellLight™ Mitochondria-GFP and loaded with 50 nM TMRM for 10 min at 37°C. (A–E) Images were acquired at 5 sec intervals for 90 sec following treatment with the uncoupler CCCP; zoomed sections (B–D) reveal heterogeneity in mitochondrial membrane potential regulation. Transient depolarization was observed in one but not all mitochondria (C, arrow), as indicated by loss of orange TMRM signal; GFP fluorescence was maintained during depolarization, indicating an intact mitochondrion. Loss of mitochondrial membrane potential was evident by 90 sec post-CCCP treatment (E); however, mitochondria were still intact, information that would have been lost using TMRM alone. 
Figure 3. Multiplex imaging of mitochondrial calcium levels and dynamics.(A) HeLa cells were labeled with CellLight™ Mitochondria-GFP and 5 μM rhod-2 AM for 15 min at 37°C before imaging live over 100 sec. (B–D) The region outlined in (A) is enlarged to show individual mitochondria within a single cell over time. (C, D) Calcium is released from internal stores following application of 10 μM histamine. Mitochondria in close proximity to the calcium release are revealed by the increase in the orange-red fluorescence of rhod-2. The arrow in (C) denotes a mitochondrion that may have impaired calcium uptake, a detail that would have been missed using rhod-2 AM alone. The asterisk marks a mitochondrion that shows a transient elevation in calcium levels. 
 Figure 4. Multiplex imaging of mitochondrial structure and function. Human osteosarcoma (U2OS) cells expressing CellLight™ Mitochondria-GFP (green) were treated with 200 μM tert-butyl hydroperoxide (TBHP, an inducer of oxidative stress) for 2 hr. A stain solution containing 5 μM CellROX™ Orange (orange) and 2 drops of NucBlue™ Live Cell Stain (blue) per mL of cell sample was applied for 30 min at 37°C. Cells were washed and imaged with Live Cell Imaging Solution using a confocal microscope. While the green-fluorescent mitochondria with normal morphology indicate healthy mitochondria, the orange-fluorescent spheroids suggest that a fraction of the mitochondria are showing signs of oxidative stress after TBHP treatment.

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