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 culture plate and subculture them every 7 days or before reaching 90% confluency and renew the medium every 3 - 4 days.</li>
	<li>Perform the experiments when cells are 60 - 80% confluent.</li>
</ol>
2. Preparation of Respirometer and Calorimeter
<ol>
	<li>Pipet 2.2 mL of DMEM into the respirometer chamber, insert the stopper fully, and adjust it with the stopper spacer tool to allow for optimal oxygen diffusion from air to medium.</li>
	<li>Stir continuously at 37 &#176;C until oxygen concentration (blue trace) and oxygen flux (red trace) are stable. Ensure the respirometer&#39;s software is programmed to account for the oxygen solubility of the medium used in the experiment. 
	NOTE: Different media have different oxygen solubility coefficients (e.g., DMEM = 0.92).</li>
	<li>After stabilization of the oxygen concentration in DMEM with the chamber open, select a stable portion of the oxygen concentration trace and calibrate it for 100% air saturation.</li>
	<li>Calibrate for 0% oxygen selecting a stable portion of the oxygen concentration trace when no oxygen is present in the chamber. Perform this step after 20 &#181;L titration of 230 mM sodium dithionite or after all oxygen is consumed by the biological sample at the end of an experiment.</li>
	<li>After the 100% air calibration of the respirometer, close the stopper and ensure that no bubbles are present in the chamber. 
	NOTE: a slight increase in oxygen flux will be detected in the closed chamber as the polarographic oxygen sensor (POS) consumes oxygen in order to measure the oxygen partial pressure.</li>
	<li>After ~10 min of the stopper being closed, confirm that the respirometer is ready for HepG2 cells by observing a stable oxygen flux. 
	NOTE: The calorimeter used here utilizes stainless steel ampules and requires one ampule to be used as a reference.</li>
	<li>Add 2.5 mL of H2O (dH2O) [equal volume as the samples (step 4)] to the reference ampule of the calorimeter and tighten the cap, using pliers if necessary. Clean the sealed ampule with airflow and slowly lower the ampule to position 1 in the reference channel of the calorimeter. Remain at position 1 until biological sample is prepared.</li>
</ol>
3. Preparation of HepG2 Cells for Respirometry and Calorimetry
<ol>
	<li>Confirm that the respirometer and calorimeter are prepared and calibrated. Warm DMEM and trypsin to 37 &#176;C in a water bath.</li>
	<li>Add DMEM to a fresh 100-mm plate and place in the incubator to allow for the equilibration of the medium for both CO2 and temperature. 
	NOTE: This medium will be used for the calorimetry experiment, so the appropriate volume depends on the number of ampules that will be used.</li>
	<li>Confirm with an inverted light microscope that the cells are at 60 - 80% confluent, then wash the 100-mm plate 2x with 10 mL of room temperature phosphate-buffered saline (PBS).</li>
	<li>Add 3 mL of the 37-&#176;C trypsin to the 100-mm plate of cells and incubate them for 7 - 10 min at 37 &#176;C in the cell culture incubator. Neutralize the trypsin with 3 mL of 37-&#176;C DMEM and suspend by gently pipetting it ~20 times with a 5 mL pipet.</li>
	<li>Transfer the 6 mL of resuspended cells in DMEM and trypsin into a sterile 15-mL conical tube and centrifuge it for 5 min at 175 x g. Remove the liquid without disturbing the cell pellet.</li>
	<li>Resuspend the cell pellet in ~5 mL of DMEM and pipet it thoroughly to ensure the cells are sufficiently dissociated from one another.</li>
	<li>Remove ~100 &#181;L of the well-mixed sample and perform a thorough cell count utilizing all 8 fields on a hemocytometer to determine the total number of cells. Then calculate the volume for resuspension of the cells in order to generate ~2 x 106 cells per 50 &#181;L. 
	NOTE: This will ensure that 2 x 106 cells are added to each chamber of the respirometer with minimal medium displacement.</li>
	<li>Centrifuge the cells for 5 min at 175 x g. Resuspend the pellet in the calculated volume of DMEM from step 3.7.</li>
	<li>Perform a cell count to determine the volume required to inject 2 x 106 cells per chamber in the respirometer (this should be ~50 &#181;L). Store the cells for the respirometric measurement on ice until ready.</li>
	<li>While the concentrated cell sample is on ice, determine the dilution required to produce a concentration of 1 x 105 cells/mL for the calorimetry.</li>
	<li>Mix the sample well and prepare ~3 mL of the cells at 1 x 105 cells/mL in DMEM for each ampule. 
	NOTE: The DMEM used for the sample dilution should be the equilibrated medium prepared in step 3.3.</li>
</ol>
4. Calorimetry
<ol>
	<li>Ensure that calorimeter is connected to the computer and the calorimetric signal in &#181;W is observable and stable.</li>
	<li>Add 2.5 mL of the cells at 1 x 105 cells/mL (~2.5 x 105 cells) to each ampule, ensuring sufficient air is between the lid of the ampule and the medium to allow for gas diffusion.</li>
	<li>Immediately tighten the cap, using pliers if necessary. Clean the sealed ampule, using airflow to remove any external debris, and slowly lower the ampule to position 1 of the calorimeter. Record the time the ampule is lowered to position 1.</li>
	<li>Allow the ampule to sit at position 1 for 15 min. 
	NOTE: During this 15 min period, cells are to be injected into the respirometer (step 5). This ensures that the same interval of time is used for both heat production and oxygen consumption when the CR ratio is determined.</li>
	<li>After 15 min at position 1, slowly lower both the reference and sample ampules to position 2. Record the time the ampules were lowered and measure for at least 30 min.</li>
</ol>
5. Respirometry
<ol>
	<li>During the 15-min interval in which the ampule is at position 1 in the calorimeter, begin the respirometric studies.</li>
	<li>Thoroughly mix the cell sample by pipetting to ensure a homogenous solution and inject &#60;50 &#181;L of the cell sample into each chamber of the respirometer for a final concentration of ~2 x 106 cells/chamber. Record the time the cells are injected into the respirometer.</li>
	<li>After the injection, the HepG2 cells are continuously stirred at 37 &#176;C. Wait at least 20 - 30 min for both a stabilization of the oxygen flux and a steady decrease in the oxygen concentration.</li>
	<li>Select the O2 flux per V tab on the right of the screen and highlight a stable portion of oxygen flux. Select marks, then statistics and record data for O2 flux per V. This recording will be used in calculation of the CR ratio. 
	NOTE: Steps 5.5 and 5.6 are optional. Leak respiration and maximal uncoupled respiration will be revealed by titrations of oligomycin and FCCP.</li>
	<li>Inject 1 &#181;L of 4mg/mL oligomycin into each chamber and allow ~ 10 min for oxygen flux stabilization. Record stable leak respiration rate by following steps performed in 5.4 by highlighting a stable portion of the oxygen flux trace after oligomycin addition.</li>
	<li>Titrate 2-&#181;L injections of 0.2 mM FCCP into each chamber, allowing for oxygen flux stabilization after each injection. Continue the titrations until the maximal flux is reached, as indicated by a slight decline in the subsequent FCCP titration. Record the stable respiration rate during the maximal flux.</li>
</ol>
6. Calculation of Calorespirometric Ratio
<ol>
	<li>Perform a final cell count of the samples prepared at ~ 1 x 105 cells/mL utilizing all 8 fields of a hemocytometer to determine the total number of cells added to the ampule (2.5 mL).</li>
	<li>Determine a time to record measurements from both calorimeter and respirometer at which&#160;stable signals are&#160;present at identical times. Reference the time recorded when the ampule was lowered to position 2 and the time of injection of cells into the respirometer.</li>
	<li>Record the heat production by placing the cursor over the trace displaying heat output for the respected ampule at the time determined in step and express as &#181;W/million cells. Collect oxygen consumption data and ensure that it is expressed as pmol O2 x&#160;s-1&#160;x&#160;million cells.</li>
	<li>To calculate the CR ratio, first convert &#181;W to kJ/s, and then divide kJ/s over mol of O2/s to obtain the CR ratio expressed as kJ/mol O2. NOTE: The CR ratio is presented in kJ/mol O2. 
	(see&#160;<a href="/files/ftp_upload/57724/JoVE_Supplemental_File.docx" target="_blank">Supplementary FIle 1</a>)</li>
</ol>

<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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+preconditioning+of+cells+with+AICAR-riboside:+improved+cryopreservation+and+cell-type+specific+impacts+on+energetics+and+proliferation.">Metabolic preconditioning of cells with AICAR-riboside: improved cryopreservation and cell-type specific impacts on energetics and proliferation.</a> Cryobiology. 61, 79-88 (2010).</li><li>Webb, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Calorimetry. , (1985).</li><li>Neven, L. G., Lehrman, N. J., 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=Effects+of+temperature+and+modified+atmospheres+on+diapausing+5th+instar+codling+moth+metabolism.">Effects of temperature and modified atmospheres on diapausing 5th instar codling moth metabolism.</a> Journal of Thermal Biology. 42, 9-14 (2014).</li><li>Brueckner, D., Solokhina, A., Krahenbuhl, S., Braissant, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+application+of+tunable+diode+laser+absorption+spectroscopy+and+isothermal+micro-calorimetry+for+calorespirometric+analysis.">A combined application of tunable diode laser absorption spectroscopy and isothermal micro-calorimetry for calorespirometric analysis.</a> Journal of Microbiological Methods. 139, 210-214 (2017).</li><li>Hasan, S. M. K., Manzocco, L., Morozova, K., Nicoli, M. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcalorimetric+measurement+of+aerobic+cell-metabolism+in+unstirred+cell-cultures.">Microcalorimetric measurement of aerobic cell-metabolism in unstirred cell-cultures.</a> Thermochimica Acta. 172, 105-113 (1990).</li></ol>

The authors have nothing to disclose.

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. Contamination by bacteria or fungi may result in misappropriated heat production or oxygen consumption, rendering the CR ratio meaningless. Further, improper cell counts, poor sample mixing, or clumped cells can also result in an inaccurate CR ratio. A critical step in regard to the respirometer is proper sample preparation so that the injection is below 50 &#181;L in volume. This volume ensures a minimal amount of medium is displaced from the chamber and the oxygen consumption is correctly attributed to 2 x 106 cells. Once routine respiration is observed, the proposed protocol is compatible with two additional titrations (oligomycin and FCCP), which illuminate leak respiration and ETS capacity of the mitochondria. For this application, excessive addition of FCCP can inhibit respiration and prevent&#160;maximal flux; therefore, at least 3 - 5 titrations should be performed prior to reaching the maximal uncoupled respiration.
Maintenance and proper calibration of both the respirometer and calorimeter is critical for calorespirometry. The titration protocol for background oxygen consumption is highly recommended and is performed with step-wise titrations of dithionite to reach decreased oxygen levels within the chamber. This is an essential measure to confirm tightly sealed chambers and correct for oxygen back diffusion into the chambers, especially if measurements are performed at a low oxygen tension. Also, performing a stirrer test before addition of cells can provide information about the responsiveness of the POS. If responsiveness is poor or the oxygen flux is not stable (Figure 2), service to the POS is required. Improper cleaning of the respirometer chambers can leave behind residual amounts of inhibitors or uncouplers, which may affect subsequent experiments. Air bubbles in the sealed chamber are also capable of interfering with the measurements and can be introduced during the closing of the chamber or during sample injection if air bubbles are present within the syringe. The calorimeter&#8217;s protocol is more direct, but it is also susceptible to experimental errors. For example, the improper sealing of the ampule will allow evaporation to occur which massively changes heat flow.
One of the limitations of this method is that not all cells can be cultured in suspension and many cell lines are adherent to the cell culture plate or substrate. In order to perform experiments, attached cells have to be enzymatically dislodged from the plate through the use of dissociation agents such as trypsin. Cells in this state may not be in the logarithmic growth phase and their physiology may be altered. Thus,&#160;subsequent analysis via the calorimeter and respirometer of recently trypsinized cells may assess disrupted cellular activities. Further, a limitation in the presented calorimetric protocol is the sedimentation of cells in the enclosed ampules, which may lead to local hypoxia around the cells due to slow oxygen diffusion in aqueous solutions. We have determined that ~3 x 105 cells is the upper limit compatible with the ampules used in this protocol. Increasing the number of cells can generate a hypoxic environment from the sedimentation of the cells, compromising the results. Thus, optimization is critical if a different cell line is investigated and alternative approaches should be considered such as increasing the medium density to reduce the sedimentation of cells24. Ampules are available that permit stirring; however, it is critical to ensure a minimal signal-to- noise ratio is present during the stirring. Additionally, calorimeters besides the one employed in this protocol are compatible with calorespirometry and their capabilities may be beyond what we have presented. Another limitation of calorespirometric analysis via a two-chamber respirometer is the inability for a high-throughput analysis, which would be most relevant for pharmaceutical development. This limitation, however, is offset by the capacity for a high-resolution characterization of the compound&#39;s influence on the respiratory chain and mitochondrial physiology.
The capacity for both high-resolution and simultaneous measurement of heat output and oxygen flux from the same sample is a significant strength of this method. A potential gold standard when using adherent cells would be to both culture and analyze cells grown on microcarrier beads in suspension. This method would avoid the negative impact of the dissociation from the cell culture surface and allow for the analysis of cellular functions in different phases of the growth curve (logarithmic phase vs. contact-inhibited phase7). Unfortunately, using microcarrier beads can be problematic due to the high stirring speed required in the respirometer and the potential for grinding of the beads along with cells underneath the stir bar. Despite these limitations, a broad range of applications remains compatible with calorespirometry. In particular, this method is poised to better facilitate pharmaceutical development by quickly identifying mitotoxic compounds in cultured cells.

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><tbody><tr><td>HepG2 Cells</td><td>American Type Culture Collection</td><td>HB-8065</td><td>Cells used for calorespirometry</td></tr><tr><td>O2k-Respirometer</td><td>Oroboros Instruments</td><td>10022-02</td><td>Respirometer</td></tr><tr><td>LKB 2277 thermal activity monitor (TAM)</td><td>Thermometric AB</td><td></td><td>Thermometric was purchased by TA Instruments</td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Thermofisher Scientific</td><td>11360070</td><td>100x solution added to DMEM medium</td></tr><tr><td>Fetal Bovine Serum - Premiuim Select</td><td>Atlanta Biologicals</td><td>S11550</td><td>Added to 10% in DMEM medium</td></tr><tr><td>Trypsn-EDTA (0.25%)</td><td>Thermofisher Scientific</td><td>25200072</td><td>Cell dissociation reagent</td></tr><tr><td>Oligomycin from &lt;em&gt;Streptomyces diastatochromogenes&lt;/em&gt;</td><td>Sigma Aldrich&amp;nbsp;</td><td>O4876</td><td>Mitochondrial Inhibitor</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td><td>Sigma Aldrich</td><td>C2920</td><td>Mitochondrial Uncoupler</td></tr><tr><td>Corning 100 mm TC-Treated Culture Dish</td><td>Corning Corporation</td><td>430167</td><td>Tissue culture dish</td></tr><tr><td>Glucose, powder</td><td>Thermofisher Scientific</td><td>15023021</td><td>Glucose for DMEM medium</td></tr><tr><td>Galactose, powder</td><td>Fischer Scientific</td><td>BP656500</td><td>Galactose for DMEM medium</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Thermofisher Scientific</td><td>25030081</td><td>Glutamine for DMEM medium</td></tr><tr><td>DMEM, no glucose</td><td>Thermofisher Scientific</td><td>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.
Proper instrumental calibration and maintenance are also critical for informative and reproducible calorespirometric measurements. By following the manufacturer&#39;s established cleaning protocol for the respirometer, extensive washes with ethanol are implemented to ensure that residual hydrophobic uncouplers and inhibitors are removed and do not compromise the measurements by partially inhibiting or uncoupling mitochondria.&#160;When the respirometer chamber is operating prior to the sample measurement, both the oxygen concentration and the oxygen flux traces should be stable as shown in Figure 1. If the POS is in need of sensor service, the oxygen flux will not stabilize as seen in Figure 2.&#160;Measurements should not be performed when the POS is not properly serviced. Additionally, the ampules utilized in the calorimeter should be cleaned, sterilized, and dried prior to use, as biological contamination can interfere with the heat production of the sample.
Cell samples are prepared after the proper calibration and preparation of both the respirometer and the calorimeter. First, the stopper in the respirometer is closed and the chamber is inspected to ensure no air bubbles are present. Then, HepG2 cells are concentrated to ~2 x 106/50 &#181;L for respirometry and immediately placed on ice. For calorimetry, HepG2 cells from the concentrated sample are diluted in equilibrated DMEM at&#160;~1 x 105 cells/mL. After cells are thoroughly mixed by pipetting, 2.5 mL of the suspension is added to a sterile ampule. The ampule is tightly sealed, and any external debris is removed by blowing the ampule with a stream of air from an air pump. The ampule is then slowly lowered to position 1, where it remains for 15 min. After 15 min has passed, both the reference and sample ampules are then slowly lowered to position 2, and measurements can begin to be recorded once the heat output is stable (Figure 3).
A final cell count is performed after the cells are added to both the calorimeter and the respirometer to determine the precise number of cells introduced into the calorimeter. The heat production is then expressed per million cells. If the HepG2 cells are cultured in the absence of glucose and galactose is supplemented to increase&#160;mitochondrial involvement, the cells do not proliferate inside the calorimeter, but instead decrease their metabolic activity after about 30 min in the ampule. This restricts the CR calculation to the earliest time point at which the heat dissipation was measured (Figure 4).
It is critical to record the time the cells are added to the ampule so that the measurements of heat production and oxygen consumption are collected at identical time points. If this precaution is not taken, important experimental errors can be introduced in the determination of the CR ratio. During the 15-min period, while the ampule is at position 1 in the calorimeter, the cells are sufficiently mixed by pipetting them, and 2 x 106 cells are injected into the respirometer via a Hamilton syringe. Oxygen flux stabilizes after approximately 15 min and oxygen concentration steadily declines (Figure 5). Again, it is critical that the time of the sample injection is recorded for data analysis. The stabilized oxygen flux of the cells serves as the surrogate for oxygen consumption when calculating the CR ratio. Additional titrations are performed to assess the leak respiration (1 &#181;L oligomycin) indicated by a drop to a lower oxygen flux and the maximal uncoupled respiration (3 - 5 titrations of 2 &#181;L FCCP) indicated by the failure to increase the respiration after successive FCCP titrations. An incomplete uncoupling or an inhibition of respiration may not be observed if the FCCP stock has expired (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57724/57724fig1.jpg" /> 
Figure 1: 100% air calibration of respirometer and status of polarographic oxygen sensor (POS). The signals for the oxygen concentration (blue line) and the oxygen flux (red line) both stabilize prior to the 100% air calibration. The stable lines indicate that the respirometer is ready for calibration. The calibration should be performed in DMEM, and not in H2O. <a href="/files/ftp_upload/57724/57724fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57724/57724fig2.jpg" /> 
Figure 2: POS in need of service. After the stabilization of the oxygen concentration (blue trace), the oxygen flux (red trace) fails to stabilize. The POS should not be used for experimentation and service should be applied as described by the manufacturer. An increased instability of the oxygen flux correlates with an increased demand for POS service. <a href="/files/ftp_upload/57724/57724fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57724/57724fig3v2.jpg" /> 
Figure 3: Approximately 2.5 x 105 HepG2 cells cultured in glucose medium show signs of proliferation in the calorimeter. Loading ~2.5 x 105 HepG2 cells cultured in the presence of glucose per ampule generates a constant heat production of -20 to -28 &#181;W using the 30-&#181;W setting on the instrument and an increased heat production over time correlates with cellular proliferation inside the ampule. <a href="/files/ftp_upload/57724/57724fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57724/57724fig4v2.jpg" /> 
Figure 4: Approximately 2.5 x 105 HepG2 cells cultured in galactose medium do not proliferate and heat production decreases over time. Loading ~2.5 x 105 HepG2 cells cultured in galactose per ampule results in an immediate heat production of ~-20 &#181;W. However, the heat production decreases over time and restricts CR-ratio measurements to the early time points of the experiment. <a href="/files/ftp_upload/57724/57724fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57724/57724fig5.jpg" /> 
Figure 5: Routine, uncoupled, and leak respiration of HepG2 cells. Once cells were injected into the respirometer, oxygen concentration steadily decreased (blue trace). After the stabilization of the oxygen flux (red trace) during the routine respiration of intact cells, oligomycin was added to inhibit the F0F1-ATP synthase. This is indicated by the stabilization of&#160;oxygen flux after oligomycin addition, which reveals leak respiration. Approximately 3 - 5 titrations of FCCP should be performed to uncouple&#160;respiration from&#160;ATP synthesis&#160;and to reveal&#160;electron transport system (ETS) capacity. <a href="/files/ftp_upload/57724/57724fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57724/57724fig6.jpg" /> 
Figure 6: Unsuccessful uncoupling of HepG2 cell respiration. After addition of cells, oxygen flux stabilized (red trace) during routine respiration and oxygen concentration steadily decreased (blue line). Titration of FCCP resulted in an incomplete uncoupling and eventual inhibition, preventing the maximal ETS capacity from being reached. FCCP was likely contaminated or degraded from long-term storage. If observed, a fresh FCCP stock must be prepared. Also, confirm that&#160;oligomycin does not inhibit&#160;ETS capacity during uncoupling. <a href="/files/ftp_upload/57724/57724fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

calorespirometry-powerful-noninvasive-approach-to-investigate

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.5K Views.  University of Louisville. Dit protocol beschrijft calorespirometry, de directe en gelijktijdige meting van zowel warmteafvoer en ademhaling, waarmee een noninvasive aanpak om te beoordelen van de energie-stofwisseling. Deze techniek wordt gebruikt ter beoordeling van de bijdrage van zowel aerobe en anaerobe emissieroutes naar energie gebruik door toezicht op de totale cellulaire energiestroom.

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

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Biology

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Immunology and Infection

Ethanol Impairs AM Bioenergetics by Shifting to Glutamine-Dependent Respiration

Assessment of Glutamine as a Fuel Source for Alveolar Macrophages Exposed to Chronic Ethanol Using an Extracellular Flux Bioanalyzer

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use impairs the ability of AMs to phagocytize and clear pathogens from the alveolar space, in part through dysregulated fuel metabolism and bioenergetics. Our prior work has shown that chronic ethanol (EtOH) consumption impairs mitochondrial bioenergetics and increases lactate levels in AMs. Further, we recently demonstrated that EtOH increases glutamine dependency and glutamine-dependent maximal respiration while decreasing flexibility, shifting away from pyruvate-dependent respiration and towards glutamine-dependent respiration. Glutaminolysis is an important compensatory pathway for mitochondrial respiration when pyruvate is used for lactic acid production or when other fuel sources are insufficient. Using a mouse AM cell line, MH-S cells, exposed to either no EtOH or EtOH (0.08%) for 72 h, we determined the dependency of mitochondrial respiration and bioenergetics on glutamine as a fuel source using an extracellular flux bioanalyzer. Real-time measures were done in response to bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), an inhibitor of glutaminase 1, which prevents the enzymatic conversion of glutamine to glutamate, in media vehicle or in response to vehicle alone, followed by testing mitochondrial stress. The step-by-step protocol provided herein describes our methods and calculations for analyzing average levels of glutamine-dependent basal mitochondrial respiration, mitochondrial ATP-linked respiration, maximal mitochondrial respiration, and mitochondrial spare respiratory capacity across multiple biological and experimental replicates.

Alcohol misuse impairs alveolar macrophage (AM) immunity due to suppressed mitochondrial respiration and bioenergetics. We recently demonstrated that ethanol (EtOH) exposure increases glutamine dependency for mitochondrial respiration in AMs. Herein, methods are provided to determine the usage of glutamine for mitochondrial respiration in EtOH-treated AMs using an extracellular flux bioanalyzer.

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer

Source: Traba, J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61466">Analysis of Human Natural Killer Cell Metabolism.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates the method to analyze energy metabolism in activated human NK cells using an extracellular flux analyzer. Different inhibitors of the electron transport chain complexes impact ATP synthesis, which is measured through the oxygen consumption rate or OCR, providing insights into cellular metabolism and bioenergetics.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Reagent preparation

	Reagents ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video