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How to Measure the Energetic Status of Cells?

Garrett Heinrich
Tags: Successful Research Tips,

Summary

  • Cells can package energy into chemical bonds for later use in a molecule called adenosine triphosphate (ATP).
  • ATP levels drop when cells are starved of nutrients or about to die.
  • Cells under energetic stress can be labeled and visualized by dyes or antibodies using microscopy or flow cytometry.


Introduction

How are you doing?
This is easy to ask your friends or colleagues, but ask some cells in a dish, and you will not get much response. So how do you know if the cells you are culturing are growing as they should be? Or if your treatment is working? Some of the greatest challenges in science are determining how to visualize cellular processes that cannot be seen with the naked eye and trusting what you see.


How do cells make energy?

Many cellular processes run on the fuel known as adenosine triphosphate (ATP), therefore the amount of ATP in a cell can be a measure of how much energy it possesses. ATP comprises a nitrogenous base (adenine), a sugar (ribose), and three phosphate groups bound to the ribose in a chain. Energy is found in the phosphate bonds and is released when the bonds are broken. Breaking a phosphate bond and removing the outer phosphate converts ATP to adenosine diphosphate (ADP), and allows the released energy to drive various cellular processes.

ATP can be produced from glucose, amino acids, or stores of fatty acids and glycogen. The process of cellular respiration, through which cells make ATP from glucose through glycolysis, the TCA cycle, and oxidative phosphorylation, has recently been described in the TechNote "What is the role of mitochondria in cellular metabolism and bioenergetics?" Amino acid and fatty acid metabolites also enter the TCA cycle and undergo oxidative phosphorylation to make ATP. Glycogenolysis converts glycogen to glucose-1-phosphate and then glucose-6-phosphate, which follows the path of glycolysis and the TCA cycle. Each of these methods ends up in the cell's mitochondria and uses electron transfer over a proton gradient to generate the energy needed for the enzyme ATP synthase to make ATP from ADP and phosphate.


What does ATP tell us?

Now that we have reviewed how ATP is made and can be used to store energy, how can we use that to infer the 'cell's energy status or overall health? Measuring the ratio of ATP to ADP (ATP/ADP or, conversely, ADP/ATP) is one way to monitor cellular energy levels. Decreasing ATP/ADP ratio is correlated with autophagy, the lysosomal-dependent process by which cellular debris is cleared from the cell and recycled. In the example of starvation, when ATP/ADP ratios are lower due to a lack of glucose intake, autophagy is induced to help cells survive. At the other end of the metabolic spectrum, a rising ATP/ADP ratio is associated with proliferating cells. The ATP/ADP ratio can also be used to screen for apoptosis and necrosis in cells as ATP decreases and ADP increases during the process. Chemiluminescence can visualize these changes with the ApoSENSOR™ ADP/ATP ratio assay kit. As apoptosis is induced, ADP/ATP ratios increase along with Caspase-3 and other apoptosis markers (Figure 1).



Figure 1. ADP/ATP ratio in Jurkat cells after apoptosis is induced by camptothecin using the ApoSENSOR™ ADP/ATP ratio assay kit.


Dannheisig et al.1 recently used the ApoSENSOR™ ADP/ATP ratio assay kit in their study to understand the role of Peter Pan (PPAN) protein, a ribosome biogenesis factor, in mitochondria. They previously showed that knockdown of PPAN results in nucleolar stress response in the mitochondria leading to apoptosis. In this work, they knocked down PPAN levels in HeLa cells using silencing RNA, measured the resulting relative ATP levels along with the ADP/ATP ratio, and found that while PPAN knockdown increased the overall ATP level, the ADP/ATP ratio was unchanged, indicating an alternative to respiration-coupled ATP production occurs to keep ATP levels high after knockdown. Following this finding, they discovered PPAN knockdown leads to disruption of the integrity of the mitochondrial membrane, decreased mitochondrial mass, and autophagy.

An alternative way to detect cell viability and apoptosis is by measuring overall ATP levels. Rapid loss of ATP is a sign of apoptosis and cell death as cellular energy stores are depleted. The ApoSENSOR™ Cell Viability Assay Kituses a bioluminescent reaction (Figure 2) to measure cellular ATP content (Figure 3).



Figure 2. Chemiluminescent reaction used by ApoSENSOR™ Cell Viability Assay Kit to determine ATP levels in cells.




Figure 3. ATP decrease as measured in Jurkat cells after camptothecin-induced apoptosis. Cell apoptosis is indicated by increased Annexin V staining.



Energy Status in Individual Cells

These assays measure ATP and ADP in whole cell populations, but having the sensitivity to detect ATP levels in individual cells is much more difficult. We do have the ability to visualize whether cells are viable or not, and whether or not they are actively proliferating. Sometimes, this can be measured in live cells by fluorescence microscopy or flow cytometry. Sometimes, cells or tissue need to be fixed and analyzed. Enzo's NUCLEAR-ID cell viability reagents come in various color combinations and can be used to stain live cells and determine if they are alive or dead by fluorescing at different wavelengths. These come in combinations of Red/Green, Blue/Green, or Blue/Red (GFP-CERTIFIED) to be compatible with any other fluorescent cell markers.

Cell proliferation can be measured by immunohistochemistry using an antibody to the nuclear protein Ki-67. Ki-67 cannot be visualized in resting cells in the G0 phase of the cell cycle, but is present in the active G1, S, G2, and M phases of dividing cells (for more information on the cell cycle, see recent TechNote, "The Cell Cycle Explained, and How to Study It"). Ki-67 has a short 1.0-1.5 hour half-life, so it can only be detected when cells are proliferating. This characteristic makes it an effective diagnostic marker for a number of tumor types and overall levels can be a prognostic indicator of future cancer progression. Enzo has Ki-67 monoclonal antibody in undiluted or ready-to-use concentrations to label proliferating cells.

Healthy cells in interphase are actively transcribing RNA to make the proteins that allow cellular processes to proceed. In this state, the nuclear DNA, organized into chromatin structures, is exposed to enable transcription factors to bind to their respective promoter regions. When cells get ready to divide, the chromatin condenses and becomes more tightly packed, taking up less space in the cell. Starving cells or cells undergoing apoptosis also experience this condensing of chromatin, and it is believed that this condensation is to slow down active transcription, preserving cellular resources in a time of stress. This morphology can be visualized with the NUCLEAR-ID® Green chromatin condensation detection kit.

As the organelle at the center of the majority of energy production (via ATP) in the cell, monitoring the overall function of the mitochondria is a useful tool in determining the energy status. The reduction of ATP generation and energy levels resulting from mitochondrial dysfunction prevents the cells from having the ability to perform other biochemical reactions and critical functions such as muscle contractions in the heart. Mitochondria also store calcium that can affect ion channel function and polarization of membrane potential. Altered polarization affects neurons and their firing frequency and is thought to have roles in neurological diseases such as 'Alzheimer's and 'Parkinson's disease, and can affect systems such as blood pressure and circadian rhythms. When caspase proteins are activated and apoptosis is induced, membrane polarization is compromised along with production of reactive oxygen species (ROS) and eventually cell death. Mitochondrial membrane potential can be determined using the MITO-ID® Membrane potential detection / cytotoxicity kits. This is a cell-based assay that uses fluorescent color change of a mitochondrial membrane potential (MMP) sensitive dye. In energized cells, the dye exists as green monomers in the cytosol and orange aggregates in the mitochondria (Figure 4, left), but in apoptotic and necrotic cells the dye leaves the mitochondria and the cell loses orange fluorescence, leaving only green (Figure 4, right). This assay can be used with either fluorescent microscopy to visualize individual cells or flow cytometry.



Figure 4. Flow cytometry of Jurkat cells untreated (left) and treated with CCCP for 15 minutes (right) using MITO-ID® membrane potential dye.


A prime example of using the MITO-ID® Membrane potential detection/cytotoxicity kits in energetics and drug toxicity studies was a recent publication on the effects of cannabis use on neuronal development. Beiersdorf et al.2 treated primary neurons derived from the cortex of prenatal mice with increasing concentrations of plant extract Δ9-tetrahydrocannabinol (THC) and visualized the mitochondrial membrane potential by fluorescence microscopy. They found that daily administration of THC lowers the mitochondrial membrane potential (MMP) in the primary neurons. They also showed that this lowered potential leads to changes in the mitochondrial proteome and contributes to growth defects. Interestingly, they also found that neuronal growth could be rescued by treatment with antioxidants and other interventions. While not used for this purpose in the publication, the authors could have also used the MITO-ID® Membrane potential detection/cytotoxicity kits to screen protective compounds based on the effects on MMP and discover more targets that may inhibit the negative effects of THC on the unborn.


How can Enzo help guide your metabolic research?

Now you have some ideas about how to measure the energy and health status of your cells, or determine if your treatment or drug candidate has any effect that would cause the cells mitochondrial dysfunction or even death. Do you have more questions on cellular energy monitoring and how to find the best assays and molecules for your research? Reach out to our Technical Support Team. We will be happy to assist!

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References:

  1. D.P. Dannheisig, et al. (2019) Loss of Peter Pan (PPAN) affects mitochondrial homeostasis and autophagic flux. Cells 8, 894. Abstract.
  2. J. Beiersdorf, et al. (2020) Adverse effects of Δ9-tetrahydrocannabinol on neuronal bioenergetics during postnatal development. JCI Insight 5, e135418. Abstract.

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