Name: calorespirometry: a powerful, noninvasive approach to investigate cellular energy metabolism
Uploaded: 2018-05-31T14:30:00
Description: Watch this Scientific Journal Video about Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism at JoVE.com

This work was funded in part by the National Science Foundation grant CHE-160944 to Mary E. Konkle and Michael A. Menze.

1. Cell Culture
<ol>
	<li>Maintain human hepatocellular carcinoma (HepG2) cells in Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) containing 10% fetal bovine serum (FBS) and additional substrates (10 mM glucose, 2 mM glutamine, and 1 mM pyruvate) at 37 &#176;C in a cell culture incubator (5% CO2, 95% air, 100% humidity). 
	NOTE: Use the above DMEM formulation when DMEM is referenced in later steps for respirometry and calorimetry.</li>
	<li>Plate the cells at 1 x 106 cells per 100 mm cultu...

<ol>
	<li>Gnaiger, E., Kemp, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+metabolism+in+aerobic+mammalian+cells:+information+from+the+ratio+of+calorimetric+heat+flux+and+respirometric+oxygen+flux.">Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux.</a> Biochimica et Biophysica Acta. 1016, 328-332 (1990).</li><li>Barros, N., Gallego, M., Feijoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+the+specific+rate+of+catabolic+activity+(Ac)+from+the+heat+flow+rate+of+soil+microbial+reactions+measured+by+calorimetry:+significance+and+applications.">Calculation of the specific rate of catabolic activity (Ac) from the heat flow rate of soil microbial reactions measured by calorimetry: significance and applications.</a> Chemistry &amp; Biodiversity. 1, 1560-1568 (2004).</li><li>Cheney, M. A., Fiorillo, R., Criddle, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbicide+and+estrogen+effects+on+the+metabolic+activity+of+Elliptio+complanata+measured+by+calorespirometry.">Herbicide and estrogen effects on the metabolic activity of Elliptio complanata measured by calorespirometry.</a> Comparative Biochemistry and Physiology - Part C: Pharmacology, Toxicology and Endocrinology. 118, 159-164 (1997).</li><li>Wadso, L., Hansen, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorespirometry+of+terrestrial+organisms+and+ecosystems.">Calorespirometry of terrestrial organisms and ecosystems.</a> Methods. 76, 11-19 (2015).</li><li>Gnaiger, E., Woakes, A. J., Grieshaber, M. K., Bridges, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+energetics+at+very+low+oxygen:+information+from+calorimetry+and+respirometry.">Animal energetics at very low oxygen: information from calorimetry and respirometry.</a> Strategies for Gas Exchange and Metabolism. , 149-171 (1991).</li><li>Barros, N., Hansen, L. D., Pineiro, V., Perez-Cruzado, C., Villanueva, M., Proupin, J., Rodriguez-Anon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+calorespirometric+ratios+of+soil+microbial+metabolism.">Factors influencing the calorespirometric ratios of soil microbial metabolism.</a> Soil Biology and Biochemistry. 92, 221-229 (2016).</li><li>Menze, M. A., Chakraborty, N., Clavenna, M., Banerjee, M., Liu, X. H., Toner, M., Hand, S. 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C., Scampicchio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ascorbic+acid+and+light+on+reactions+in+fresh-cut+apples+by+microcalorimetry.">Effects of ascorbic acid and light on reactions in fresh-cut apples by microcalorimetry.</a> Thermochimica Acta. 649, 63-68 (2017).</li><li>Criddle, R. S., Fontana, A. J., Rank, D. R., Paige, D., Hansen, L. D., Breidenbach, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+metabolic+heat+rate,+CO2+production,+and+O2+consumption+by+microcalorimetry.">Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry.</a> Analytical Biochemistry. 194, 413-417 (1991).</li><li>Criddle, R. S., Breidenbach, R. W., Rank, D. R., Hopkin, M. S., Hansen, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+calorimetric+and+respirometric+measurements+on+plant-tissues.">Simultaneous calorimetric and respirometric measurements on plant-tissues.</a> Thermochimica Acta. 172, 213-221 (1990).</li><li>Pesta, D., Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+respirometry:+OXPHOS+protocols+for+human+cells+and+permeabilized+fibers+from+small+biopsies+of+human+muscle.">High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle.</a> Methods in Molecular Biology. 810, 25-58 (2012).</li><li>Grimm, D., Altamirano, L., Paudel, S., Welker, L., Konkle, M. E., Chakraborty, N., Menze, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+cellular+energetics+by+galactose+and+pioglitazone.">Modulation of cellular energetics by galactose and pioglitazone.</a> Cell and Tissue Research. , (2017).</li><li>Hansen, L. D., Macfarlane, C., McKinnon, N., Smith, B. N., Criddle, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+calorespirometric+ratios,+heat+per+CO2+and+heat+per+O2,+to+quantify+metabolic+paths+and+energetics+of+growing+cells.">Use of calorespirometric ratios, heat per CO2 and heat per O2, to quantify metabolic paths and energetics of growing cells.</a> Thermochimica Acta. 422, 55-61 (2004).</li><li>Chinet, A., Clausen, T., Girardier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcalorimetric+determination+of+energy+expenditure+due+to+active+sodium-potassium+transport+in+the+soleus+muscle+and+brown+adipose+tissue+of+the+rat.">Microcalorimetric determination of energy expenditure due to active sodium-potassium transport in the soleus muscle and brown adipose tissue of the rat.</a> The Journal of Physiology. 265, 43-61 (1977).</li><li>Paul, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+biochemical+energy+balance+during+an+isometric+tetanus+and+steady+state+recovery+in+frog+sartorius+at+0+degree+C.">Physical and biochemical energy balance during an isometric tetanus and steady state recovery in frog sartorius at 0 degree C.</a> Journal of General Physiology. 81, 337-354 (1983).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+cells.">On the origin of cancer cells.</a> Science. 123, 309-314 (1956).</li><li>Kemp, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+calorimetric-respirometric+ratio+in+studying+intermediary+metabolism+of+cultured+mammalian+cells.">Importance of the calorimetric-respirometric ratio in studying intermediary metabolism of cultured mammalian cells.</a> Thermochimica Acta. 172, 61-73 (1990).</li><li>Kola, I., Landis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+the+pharmaceutical+industry+reduce+attrition+rates?.">Can the pharmaceutical industry reduce attrition rates?.</a> Nature Reviews Drug Discovery. 3, 711-715 (2004).</li><li>Kamalian, L., Chadwick, A. E., Bayliss, M., French, N. S., Monshouwer, M., Snoeys, J., Park, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+HepG2+cells+to+identify+direct+mitochondrial+dysfunction+in+the+absence+of+cell+death.">The utility of HepG2 cells to identify direct mitochondrial dysfunction in the absence of cell death.</a> Toxicology in Vitro. 29, 732-740 (2015).</li><li>Rossignol, R., Gilkerson, R., Aggeler, R., Yamagata, K., Remington, S. J., Capaldi, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+substrate+modulates+mitochondrial+structure+and+oxidative+capacity+in+cancer+cells.">Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells.</a> Cancer Research. 64, 985-993 (2004).</li><li>Fontana, A. J., Hansen, L. D., Breidenbach, R. W., Criddle, R. 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The objective of calorespirometry is to quantitatively evaluate the contributions of aerobic and anaerobic pathways to metabolic activity and obtain a composite view of cellular energy flux. This is accomplished by a simultaneous measurement of heat dissipation and oxygen flux followed by a comparison of the calculated CR ratio with the theoretical oxycaloric equivalent. Several critical steps must be considered for reproducible and reliable data. The maintenance of a sterile, healthy cell culture is critical. Contaminat...

In biological systems, the heat-release or enthalpy change during metabolism is typically monitored either directly (via direct calorimetry) or indirectly via O2 consumption and/or CO2 production (via respirometry). Unfortunately, when these techniques are used in isolation, critical information is lost, such as the contribution of anaerobic pathways to cellular metabolism. Calorespirometry is a powerful technique that relies on the concurrent measurement of both heat dissipation and respiration. Pioneering calorespirometric work investigated the anaerobic metabolism in fully oxygenated mammalian cells and demonstrated simultaneous contributions of both aerobic and anaerobic pathways to energy homeostasis despite the transformed cells being in a fully oxygenated environment1. Calorespirometry has since been applied to a wide variety of biological questions. Some examples include the study of animal energetics at low oxygen levels, the effects of both herbicide and estrogen on the gills of bivalves, the metabolism of terrestrial organisms, and the microbial decomposition of organic soil matter2,3,4,5,6. Furthermore, calorespirometry has revealed how metabolic preconditioning prior to freezing improves the cryopreservation of mammalian cells7. Each approach, both calorimetry and respirometry, has independently increased our knowledge of cellular and organismal bioenergetics. However, fundamental biological questions that can be answered through the use of calorespirometry remain relatively unexplored.
Hess&#39;s law states that the total enthalpy change of a reaction is independent of the pathway between the initial and final states. For example, the total enthalpy change for a biochemical pathway is the summation of the change in enthalpies of all reactions within the pathway. Calorimetry offers a real-time approach for measuring cellular heat production, which indiscriminately detects both aerobic and anaerobic pathways. This&#160;is based on the foundation that no energy is exchanged in the system except through the walls of the experimental ampule8. A change in heat dissipation is equal to the change in enthalpy released from all metabolic reactions in the ampule. Thus, a negative enthalpy correlates to a loss of heat from the system. Exhaustive research over the last four decades has characterized the thermodynamic landscape of both catabolism and anabolism. This is represented by a steady rise in research articles found under the search terms &#34;biological&#34; and &#34;calorimetry&#34; as indexed by the United States National Library of Medicine (NLM) at the National Institutes of Health (PubMed). The search reveals that prior to 1970, a total of 27 publications reference biological calorimetry; meanwhile, in 2016 alone, 546 publications utilized the technique.
Calorimetric methods are well established to determine heat production. However, more flexibility is granted for resolving the respirometric value. The respirometric measurement can consist of O2, CO2, or both O2 and CO2. Further, the measurement of O2 or CO2 can be accomplished by various techniques, including optrodes, Clark-type electrodes, and tunable diode laser absorption spectroscopy7,9,10,11. While CO2 production is a&#160;valuable metric in many respirometric studies, the medium for cultured cells often utilizes a bicarbonic buffer system for pH control12,13. To avoid complications of CO2 measurement in the bicarbonate system, the following protocol for the calorespirometry of cells in culture utilizes O2 as the sole respirometric parameter.
Concurrent with measuring oxygen flux, certain respirometers (see Table of Materials) are designed for detailed assessments of mitochondrial function. Substrate-uncoupler-inhibitor-titrations (SUIT) protocols are well established and are compatible with experiments designed to measure membrane potential or reactive oxygen species (ROS) formation14. The presented protocol for calorespirometry of intact cells is compatible with the introduction of chemical uncouplers such as carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the F0F1-ATP synthase inhibitor oligomycin. Through the addition of FCCP, oxygen consumption can be uncoupled from ATP production, which is useful to assess&#160;the impact of potential therapeutics on mitochondrial peformance15. Furthermore, the addition of oligomycin illuminates the extent of leak respiration. Thus, the respirometric measurements performed during calorespirometry are compatible with extensive protocols designed to further elucidate mitochondrial physiology.
The simultaneous measurement of both heat dissipation and oxygen flux allows for the calculation of the calorespirometric (CR) ratio. This ratio is then compared to the Thornton&#39;s constant or the theoretical oxycaloric equivalent, which ranges between -430 to -480 kJ mol-1 depending on the cell line or tissue of interest and the supplemented carbon substrates1,16. Thus, a more negative CR ratio reveals increased contributions from anaerobic pathways to overall metabolic activity. For example, the CR ratio for routine muscle tissue respiration without the active performance of work ranges from -448 to -468 kJ mol-1 which is within the range of the theoretical oxycaloric equivalent17,18. Meanwhile, mammalian cancer cells cultured in medium that is high in glucose display enhanced lactic acid fermentation following glycolysis and relatively low mitochondrial engagement19. This phenotype results in CR ratios in the range of -490 to -800 kJ mol-1, demonstrating a heightened involvement of the anaerobic pathways in the cellular metabolism as indicated by more negative CR ratios1,7,16,20.
Both commercial and non-profit cell and tissue distributors currently offer cell lines from over 150 species, with nearly 4,000 cell lines derived from humans. Immortalized cell lines are convenient tools for quickly evaluating the toxicity of potential therapeutics, many of which may directly or indirectly interfere with mitochondrial functions. Using transformed cells during drug screening may be of limited predictive value in part because of the Warburg effect, a hallmark of many cancers. Often, cancers generate ATP from substrate-level phosphorylation and maintain redox balance through the production of lactate without fully engaging the mitochondrion under aerobic conditions19. Pharmaceutical development is notoriously costly and inefficient, with approximately 8 out of 9 compounds tested in human clinical trials failing to achieve market approval21. While potential therapeutics may pass initial screening due to low cytotoxicity in cell lines, it is possible that some of these compounds are mitotoxic. Without a suitable method to detect how these toxins can impair the energy balance in primary cells that do not display the Warburg effect, critical information is often over-looked, bottlenecking therapeutic development in early stages.
Calorespirometry is a practical, noninvasive approach to analyze metabolic activity in a variety of biological samples, including cells and tissues. The core of the presented protocol is compatible with a range of applications. One complication, however, has been identified. Immortalized cells are often cultured in a glucose-free medium supplemented with galactose to increase the contribution of oxidative phosphorylation (OXPHOS) for energy production, in order to sensitize the cells to potential mitotoxins22,23. This metabolic reprogramming appears to obscure analysis when samples are placed in the stainless steel ampules used by the calorimeter15. Cells cultured in a glucose medium continue to engage in high metabolic activity for several hours. Meanwhile, cells cultured in galactose medium decrease the heat production within 30 min of their placement in the ampule, making measurements restricted to early experimental time points. This behavior, unfortunately, hinders the opportunity to assess their cellular proliferation. Despite this specific limitation, most applications are compatible with calorespirometric analysis and detailed metabolic information can be obtained through this approach.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HepG2 Cells</td>
			<td style="width:125px;">American Type Culture Collection</td>
			<td style="width:119px;">HB-8065</td>
			<td style="width:167px;">Cells used for calorespirometry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">O2k-Respirometer</td>
			<td style="width:125px;">Oroboros Instruments</td>
			<td style="width:119px;">10022-02</td>
			<td>Respirometer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LKB 2277 thermal activity monitor (TAM)</td>
			<td style="width:125px;">Thermometric AB</td>
			<td style="width:119px;"></td>
			<td>Thermometric was purchased by TA Instruments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Pyruvate (100 mM)</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">11360070</td>
			<td>100x solution added to DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum - Premiuim Select</td>
			<td style="width:125px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11550</td>
			<td>Added to 10% in DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsn-EDTA (0.25%)</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">25200072</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligomycin from Streptomyces diastatochromogenes</td>
			<td style="width:125px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">O4876</td>
			<td>Mitochondrial Inhibitor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td style="width:125px;">Sigma Aldrich</td>
			<td style="width:119px;">C2920</td>
			<td>Mitochondrial Uncoupler</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 100 mm TC-Treated Culture Dish</td>
			<td style="width:125px;">Corning Corporation</td>
			<td style="width:119px;">430167</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glucose, powder</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">15023021</td>
			<td>Glucose for DMEM medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Galactose, powder</td>
			<td style="width:125px;">Fischer Scientific</td>
			<td style="width:119px;">BP656500</td>
			<td>Galactose for DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-Glutamine (200 mM)</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">25030081</td>
			<td>Glutamine for DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, no glucose</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">11966025</td>
			<td>Cell culture medium</td>
		</tr>
	</tbody>
</table>

calorespirometry: a powerful, noninvasive approach to investigate cellular energy metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

The reproducibility of calorespirometric measurements depends on a proper and consistent sample preparation. Samples prepared from cell culture should not be used if the plates are overgrown, as cell counts can become inaccurate due to clumping. Further, reduced heat flows due to limited substrate diffusion in the clumped cells may occur. Therefore, when using adherent cells, it is critical to select a plate with the confluency between 60 - 80% and to change the medium 24 h prior to the experiment.
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Watch this Scientific Journal Video about Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism at JoVE.com

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

The overall goals of this procedure are to analyze the metabolic activity of a biological sample and to determine the extent that anaerobic pathways contribute to the cellular energy production within the sample. This method can help answer key questions in the pharmaceutical field about the extent of mitochondrial toxicity during drug development. The main advantage of this technique is that it facilitates the simultaneous measurement of oxygen consumption and heat production in a cell sample of interest.So this method can provide insight into treatment planning during pharmaceutical development. It can also be applied to other fields, such as cyrobiology in which the effects of metabolic preconditioning on cryopreservation can be assessed. To prepare the respirometer, first add two-point-two milliliters of DMEM to each respirometer chamber and fully insert the stoppers.Adjust the stoppers with a stopper-spacer tool to allow optimal oxygen diffusion to the medium, and set the chambers to continuously stir the samples at 750 rpm at 37 degrees Celsius until the oxygen concentration and the oxygen flux are stable. Confirm that the respirometer software is programmed to account for the oxygen solubility of the medium used in the experiment. Then select a stable portion of the oxygen concentration trace and calibrate this portion for 100%air saturation.Now, close the stoppers, taking care that no bubbles are present in the chambers. After about 10 minutes, a stable oxygen flux should be observed. To prepare for calorimetry, first add two-point-five milliliters of de-ionized water to the reference ampule, then tighten the cap.Using an air pump, remove external debris from the ampule, then slowly lower the ampule to position one in the reference channel of the calorimeter. The ampule will remain at position one until the ampule with the cell sample has been prepared. DMEM, used for calorimetry, should be equilibrated in a 37-degree Celsius incubator with a humidified atmosphere and 5%CO2.Begin the experiment by adding three milliliters of DMEM to a 100-milliliter plate for each calorimetric ampule that will be used, and place the plate in the incubator until the sample is ready for calorimetry. While the medium is equilibrating, use an inverted microscope to select a plate with HepG2 cells that are between 60 and 80%confluent and are homogeneously distributed. First, remove the cell culture medium and wash the cells two times with 10 milliliters of PBS.Add three milliliters of pre-warmed trypsin at 37 degrees Celsius and incubate the plate at 37 degrees Celsius. After seven to 10 minutes of incubation, neutralize the enzymatic reaction with the three milliliters of 37-degrees Celsius DMEM and use a five-milliliter pipette to gently dissociate the cells into a single-cell suspension. Transfer the cells into a sterile 15-milliliter conical tube for centrifugation, and re-suspend the palette in approximately five milliliters of fresh DMEM.Pipette the cells thoroughly to disaggregate any cell clusters, and after counting, concentrate the cells to approximately two millions cells per 50 microliters of medium. The reproducibility of this method is dependant upon on consistent and accurate cell counts. It is critical to ensure the sample is homogeneous and that cells are not clumped at any point during the analysis.Then, store the cells on ice until ready to perform respirometric and calorimetric measurements. Before the calorimetric measurement, confirm that the calorimeter is connected to the computer and that the calorimetric signal is stable and in micro-watts. Dilute the cells to 100, 000 cells per milliliter in the equilibrated medium that was placed in the incubator prior to sample preparation.Then add two-point-five milliliters of cell solution to each ampule, confirming that a sufficient volume of air is maintained between the lids of the ampules and the medium to allow for gas diffusion. Immediately tighten the caps and clean the sealed ampules with air to remove any external debris. Then slowly lower the sealed ampule with the cell sample to position one of the calorimeter.Thoroughly mix the sample to ensure a homogeneous solution, and inject two million cells into each closed chamber of the respirometer under continuous stirring. After 15 minutes at position one in the calorimeter, slowly lower both the reference ampule and the ampule with the cells to position two and record the time of lowering. 10 to 15 minutes after the cells have been injected into the respirometer, a stabilization of the oxygen flux and a steady decrease in the oxygen concentration should be observed.To determine leak respiration, inject one microliter of four milligrams per milliliter oligomycin into each chamber and allow the oxygen flux to stabilize for about 10 minutes, recording the stable leak respiration rate as just demonstrated. Then, to determine the maximal flux, inject two microliters of zero-point-two millimolar FCCP into the chamber, allowing the oxygen flux to stabilize after each injection, and continuing the titrations until the maximal flux is reached. Record the stable respiration rate during the maximal flux.When the respirometer chamber is operating prior to sample measurement, both the oxygen concentration and oxygen flux traces should be stable. After the ampule has been lowered to position two, a time period can be identified in which the heat output is stable, and a corresponding time can be identified in which the oxygen flux is stable, to allow calculation of the calorespirometric ratio. If the HepG2 cells are cultured in the absence of glucose and galactose is supplemented to increase the mitochondrial involvement, the cells do not proliferate inside the calorimeter, but instead decrease their metabolic activity after about 30 minutes in the ampule, restricting the calorespirometric calculation to the earliest time point at which the heat dissipation was measured.It is critical to record the time the cells are pipetted into the ampule so that the measurements of the heat production and the oxygen consumption are collected at identical time points, as it takes approximately 15 minutes for the oxygen flux to stabilize and the oxygen concentration to steadily decline. As the stabilized oxygen flux of the cells serves as a surrogate for the oxygen consumption when calculating the calorespirometric ratio, additional titrations can be performed to assess the leak respiration, which is indicated by a drop in the oxygen flux and the maximal uncoupled respiration, which is indicated by a failure to increase the respiration with successive FCCP titrations. Once mastered, this technique can be completed in less than four hours if it is performed properly.While attempting this procedure, it is important to remember that other cell lines may have different metabolic properties. Therefore, each new cell line must be optimized for both it's calorimetric and respirometric measurements. After watching this video, you should have a good understanding of how to prepare HepG2 cells for calorespirometry to ensure correct instrumental calibration, and to properly conduct a calorespirometric experiment.

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