Name: Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis (Video) | JoVE Journal
Uploaded: 2019-03-19T13:30:00.000+00:00
Duration: 8 min 48 s
Description: Learn to measure mitochondrial respiration and glycolytic function in Candida albicans using real-time extracellular flux analysis without mitochondrial purification. This method enables metabolic studies of fungal pathogens.

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5 minutes

Research in NC lab is supported by a National Institutes of Health (NIH) grant R01AI24499 and a New Jersey Health Foundation (NJHF) grant, #PC40-18.

NOTE: The detailed stepwise protocol of the assay is described below, and the schematic protocol is shown in Figure 1.
1. C. albicans strains and growth conditions
<ol>
	<li>Grow the C. albicans strains&#160;in liquid Yeast Extract-Peptone-Dextrose (YPD) medium at 30 &#176;C in an incubator shaker overnight. 
	NOTE: Maintain Candida strains as frozen stocks and grow on YPD agar (1 % yeast extract, 2% peptone, 2% dextrose, and 2% agar).</li>
</ol>
2. Preparation of reagents
<ol>
	<li>Prepare the assay medium as follows:
	<ol>
		<li>For mitochondrial function assay, dissolve 1.04 g Roswell Park Memorial Institute (RPMI) 1640 powder and 2 g glucose (2%) in 90 mL sterile water and warm the media to 37 &#176;C. Adjust the pH to 7.4 using 5 M NaOH and make up the volume to 100 mL with sterile water.</li>
		<li>For glycolytic stress assay, dissolve 1.04 g RPMI 1640 powder alone in 90 mL sterile water, warm the media to 37 &#176;C and adjust the pH to 7.4. Make up the volume to 100 mL with sterile water. RPMI 1640 powder has no bicarbonate, which is critical to monitor the pH change as a measure of glycolysis during the assay.</li>
	</ol>
	</li>
	<li>Injection compounds.
	<ol>
		<li>Prepare 1 M glucose stock in sterile water and store at -20 &#176;C.</li>
		<li>Prepare 100 mM oligomycin stock in dimethyl sulfoxide (DMSO), aliquot in small volumes and store at -20 &#176;C.</li>
		<li>Prepare 100 mM antimycin A stock in DMSO, aliquot in small volumes and store at -20 &#176;C.</li>
		<li>Prepare 100 mM SHAM (salicylhydroxamic acid) stock in ethanol on the day of assay.</li>
		<li>Prepare 1 M KCN in the sterile water on the day of the assay.</li>
	</ol>
	</li>
</ol>
3. Coating of the assay plate with Poly-D-Lysine (PDL)
NOTE: Perform all the below steps in a laminar hood.
<ol>
	<li>Dissolve Poly-D Lysine in tissue culture grade water to make 50 &#181;g/mL final concentration. Mix it well and aliquot into a 1.5 mL microcentrifuge tubes and store at -20 &#176;C for the long term. 
	NOTE: 50 &#181;L per well is needed, and for 24 wells, 1.2 mL is required. Therefore, aliquot at least 1.3 mL per microcentrifuge tube.</li>
	<li>Add 50 &#181;L per well and incubate at room temperature with the lid covered for 1-2 h.</li>
	<li>Aspirate the solution and rinse one time with 500 &#181;L sterile tissue culture grade water.</li>
	<li>Open the lid and allow the wells to air dry. Use the plate on the same day or store at 4 &#176;C for a maximum of 2-3 days.</li>
</ol>
4. Hydration of sensor cartridge
NOTE: Perform this step one day before the experiment.
<ol>
	<li>Open the extra flux Assay Kit and remove the contents. Place the sensor cartridge upside down next to the utility plate (Figure 2).</li>
	<li>Fill each well of the utility plate with 1 mL of calibrant and place the sensor cartridge back. Make sure the sensors which contain fluorophores (to measure the oxygen and pH) are submerged in the calibrant.</li>
	<li>Incubate the sensor cartridge overnight in a non-CO2 incubator at 37 &#176;C.</li>
</ol>
5. Growing and seeding cells in the PDL-coated plates
<ol>
	<li>Inoculate C. albicans in the YPD broth and grow overnight at 30 &#176;C in a shaker at 200 rpm. 
	NOTE: Based on the assay design and the interest, C. albicans can also be grown in YPG or minimal medium.</li>
	<li>On the day of assay, dilute an appropriate number of cells in the assay medium to yield a final concentration of 100,000 cells per 100 &#181;L.</li>
	<li>Add 100 &#181;L of the diluted cells into each well of the assay plate except wells A1, B4, C3, and D6, in which add only 100 &#181;L of the assay medium for background correction (Figure 3).</li>
	<li>Transfer the plate to a non-CO2 incubator at 37 &#176;C and incubate for 60 min, which will let the cells adhere to the plate surface.</li>
</ol>
6. Assay Protocol
NOTE: The protocol outlined here is for the 24-well format of the instrument. Volumes will need to be adjusted if another format is used.
<ol>
	<li>Mitochondrial function assay
	<ol>
		<li>Preparation of compounds
		<ol>
			<li>Prepare compounds at 10x concentration for mitochondrial function assay: Prepare 20 mM SHAM, 100 &#181;M Oligomycin, 100 mM KCN, and 20 &#181;M Antimycin A in the corresponding assay medium.</li>
			<li>Add 50 &#181;L SHAM into the port A, 55 &#181;L Oligomycin into port B, 62 &#181;L KCN into port C and 68 &#181;L Antimycin A into port D (Figure 4).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Glycolytic stress assay
	<ol>
		<li>Preparation of compounds
		<ol>
			<li>Prepare compounds at 10x concentration for glycolytic stress assay. Prepare 100 mM glucose, 100 &#181;M Oligomycin, 500 mM 2-Deoxy Glucose (2DG) and 20 &#181;M Antimycin A in the corresponding assay medium.</li>
			<li>Add 50 &#181;L glucose into port A, 55 &#181;L Oligomycin into port B, 62 &#181;L 2-DG into port C and 68 &#181;L Antimycin A into port D.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Employ extra flux analyzer, which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 24-well plate format. Set up the assay protocol in advance.</li>
	<li>Open the extra flux analyzer and set up the assay template by using the assay wizard tab and follow step by step instruction to fill out all the information that pops out during the setup. Generate the group layout as similar to shown in Figure 5. Set up the protocol as shown in Table 1. Set up these layouts well ahead before assay and save in the computer. At the time of assay, restore the saved protocol by opening the corresponding file in the open file option in the assay wizard tab (Figure 5).</li>
	<li>Load the 10x compounds in the respective ports of the hydrated sensor cartridge containing the calibrant and load in the carrier tray of the extra flux analyzer. Start the calibration by pressing the Start button on the screen.</li>
	<li>Add 350 &#181;L of the assay medium gently to the cell plate along the side of the wells to minimize cell disturbance to bring the final volume to 450 &#181;L.</li>
	<li>Replace the utility plate containing the calibrant with the assay plate and continue.</li>
	<li>Remove the sensor cartridge and the plate once the assay is completed. Save the file in the appropriate destination folder.</li>
</ol>
7. Data Analysis
<ol>
	<li>Use the area under the curve- analysis of variance (AUC-ANOVA) analysis tab in the software to calculate the significant difference between the groups by selecting the respective parameters (OCR or ECAR) as shown in Figure 6.</li>
	<li>Select the groups that need to be compared.</li>
	<li>Add to the analysis groups for ANOVA and click Ok. 
	NOTE: This AUC ANOVA analysis will add a new sheet to the file in which the AUC is calculated for each group and compared between them by ANOVA. This will give a table of p values to show the significance.</li>
</ol>

<ol><li>Brown, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hidden+killers%3A+human+fungal+infections%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Hidden killers: human fungal infections.</a> Science Translational Medicine. 4 (165), (2012).</li>
<li>Wisplinghoff, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nosocomial+bloodstream+infections+in+US+hospitals%3A+analysis+of+24%2C179+cases+from+a+prospective+nationwide+surveillance+study%5BTitle%5D)+AND+%22Clinical+Infectious+Diseases%22%5BJournal%5D)">Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.</a> Clinical Infectious Diseases. 39 (3), 309-317 (2004).</li>
<li>Ascioglu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+opportunistic+invasive+fungal+infections+in+immunocompromised+patients+with+cancer+and+hematopoietic+stem+cell+transplants%3A+an+international+consensus%5BTitle%5D)+AND+%22Clinical+Infectious+Diseases%22%5BJournal%5D)">Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus.</a> Clinical Infectious Diseases. 34 (1), 7-14 (2002).</li>
<li>Stover, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nosocomial+infection+rates+in+US+children%27s+hospitals%27+neonatal+and+pediatric+intensive+care+units%5BTitle%5D)+AND+%22American+Journal+of+Infection+Control%22%5BJournal%5D)">Nosocomial infection rates in US children's hospitals' neonatal and pediatric intensive care units.</a> American Journal of Infection Control. 29 (3), 152-157 (2001).</li>
<li>Wilson, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+direct+cost+and+incidence+of+systemic+fungal+infections%5BTitle%5D)+AND+%22Value+in+Health%22%5BJournal%5D)">The direct cost and incidence of systemic fungal infections.</a> Value in Health. 5 (1), 26-34 (2002).</li>
<li>Wenzel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nosocomial+candidemia%3A+risk+factors+and+attributable+mortality%5BTitle%5D)+AND+%22Clinical+Infectious+Diseases%22%5BJournal%5D)">Nosocomial candidemia: risk factors and attributable mortality.</a> Clinical Infectious Diseases. 20 (6), 1531-1534 (1995).</li>
<li>Wisplinghoff, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nosocomial+bloodstream+infections+in+pediatric+patients+in+United+States+hospitals%3A+epidemiology%2C+clinical+features+and+susceptibilities%5BTitle%5D)+AND+%22Pediatric+Infectious+Disease+Journal%22%5BJournal%5D)">Nosocomial bloodstream infections in pediatric patients in United States hospitals: epidemiology, clinical features and susceptibilities.</a> Pediatric Infectious Disease Journal. 22 (8), 686-691 (2003).</li>
<li>Cheng, W. C., Leach, K. M., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+death+pathways+in+yeast+and+mammalian+cells%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Mitochondrial death pathways in yeast and mammalian cells.</a> Biochimica et Biophysica Acta. 1783 (7), 1272-1279 (2008).</li>
<li>Shingu-Vazquez, M., Traven, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+and+fungal+pathogenesis%3A+drug+tolerance%2C+virulence%2C+and+potential+for+antifungal+therapy%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Mitochondria and fungal pathogenesis: drug tolerance, virulence, and potential for antifungal therapy.</a> Eukaryotic Cell. 10 (11), 1376-1383 (2011).</li>
<li>Brown, A. J., Brown, G. D., Netea, M. G., Gow, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolism+impacts+upon+Candida+immunogenicity+and+pathogenicity+at+multiple+levels%5BTitle%5D)+AND+%22Trends+in+Microbiology%22%5BJournal%5D)">Metabolism impacts upon Candida immunogenicity and pathogenicity at multiple levels.</a> Trends in Microbiology. 22 (11), 614-622 (2014).</li>
<li>Tucey, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+Homeostasis+Is+Important+for+Immune+Cell+Viability+during+Candida+Challenge+and+Host+Survival+of+Systemic+Fungal+Infection%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">Glucose Homeostasis Is Important for Immune Cell Viability during Candida Challenge and Host Survival of Systemic Fungal Infection.</a> Cell Metabolism. 27 (5), 988-1006 (2018).</li>
<li>Barelle, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Niche-specific+regulation+of+central+metabolic+pathways+in+a+fungal+pathogen%5BTitle%5D)+AND+%22Cellular+Microbiology%22%5BJournal%5D)">Niche-specific regulation of central metabolic pathways in a fungal pathogen.</a> Cellular Microbiology. 8 (6), 961-971 (2006).</li>
<li>Carman, A. J., Vylkova, S., Lorenz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+acetyl+coenzyme+A+synthesis+and+breakdown+in+alternative+carbon+source+utilization+in+Candida+albicans%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Role of acetyl coenzyme A synthesis and breakdown in alternative carbon source utilization in Candida albicans.</a> Eukaryotic Cell. 7 (10), 1733-1741 (2008).</li>
<li>Fradin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Granulocytes+govern+the+transcriptional+response%2C+morphology+and+proliferation+of+Candida+albicans+in+human+blood%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood.</a> Molecular Microbiology. 56 (2), 397-415 (2005).</li>
<li>Lorenz, M. C., Bender, J. A., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+response+of+Candida+albicans+upon+internalization+by+macrophages%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Transcriptional response of Candida albicans upon internalization by macrophages.</a> Eukaryotic Cell. 3 (5), 1076-1087 (2004).</li>
<li>Ramirez, M. A., Lorenz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+in+alternative+carbon+utilization+pathways+in+Candida+albicans+attenuate+virulence+and+confer+pleiotropic+phenotypes%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Mutations in alternative carbon utilization pathways in Candida albicans attenuate virulence and confer pleiotropic phenotypes.</a> Eukaryotic Cell. 6 (2), 280-290 (2007).</li>
<li>Bambach, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Goa1p+of+Candida+albicans+localizes+to+the+mitochondria+during+stress+and+is+required+for+mitochondrial+function+and+virulence%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Goa1p of Candida albicans localizes to the mitochondria during stress and is required for mitochondrial function and virulence.</a> Eukaryotic Cell. 8 (11), 1706-1720 (2009).</li>
<li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzymatic+dysfunction+of+mitochondrial+complex+I+of+the+Candida+albicans+goa1+mutant+is+associated+with+increased+reactive+oxidants+and+cell+death%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Enzymatic dysfunction of mitochondrial complex I of the Candida albicans goa1 mutant is associated with increased reactive oxidants and cell death.</a> Eukaryotic Cell. 10 (5), 672-682 (2011).</li>
<li>Desai, C., Mavrianos, J., Chauhan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+albicans+SRR1%2C+a+putative+two-component+response+regulator+gene%2C+is+required+for+stress+adaptation%2C+morphogenesis%2C+and+virulence%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Candida albicans SRR1, a putative two-component response regulator gene, is required for stress adaptation, morphogenesis, and virulence.</a> Eukaryotic Cell. 10 (10), 1370-1374 (2011).</li>
<li>Mavrianos, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+two-component+signaling+systems+in+Candida+albicans%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Mitochondrial two-component signaling systems in Candida albicans.</a> Eukaryotic Cell. 12 (6), 913-922 (2013).</li>
<li>Koch, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Mitochondrial+GTPase+Gem1+Contributes+to+the+Cell+Wall+Stress+Response+and+Invasive+Growth+of+Candida+albicans%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">The Mitochondrial GTPase Gem1 Contributes to the Cell Wall Stress Response and Invasive Growth of Candida albicans.</a> Frontiers in Microbiology. 8, 2555(2017).</li>
<li>Qu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+sorting+and+assembly+machinery+subunit+Sam37+in+Candida+albicans%3A+insight+into+the+roles+of+mitochondria+in+fitness%2C+cell+wall+integrity%2C+and+virulence%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Mitochondrial sorting and assembly machinery subunit Sam37 in Candida albicans: insight into the roles of mitochondria in fitness, cell wall integrity, and virulence.</a> Eukaryotic Cell. 11 (4), 532-544 (2012).</li>
<li>Brandt, M. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBrandt%2C+ME+%22Candida+and+Candidiasis%22">Candida and Candidiasis</a>. , American Society for Microbiology Press. book review (2002).</li>
<li>Huh, W. K., Kang, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+cloning+and+functional+expression+of+alternative+oxidase+from+Candida+albicans%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Molecular cloning and functional expression of alternative oxidase from Candida albicans.</a> Journal of Bacteriology. 181 (13), 4098-4102 (1999).</li>
<li>Yan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+alternative+oxidase+of+Candida+albicans+causes+reduced+fluconazole+susceptibility%5BTitle%5D)+AND+%22Journal+of+Antimicrobial+Chemotherapy%22%5BJournal%5D)">The alternative oxidase of Candida albicans causes reduced fluconazole susceptibility.</a> Journal of Antimicrobial Chemotherapy. 64 (4), 764-773 (2009).</li>
<li>de Moura, M. B., Van Houten, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioenergetic+analysis+of+intact+mammalian+cells+using+the+Seahorse+XF24+Extracellular+Flux+analyzer+and+a+luciferase+ATP+assay%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Bioenergetic analysis of intact mammalian cells using the Seahorse XF24 Extracellular Flux analyzer and a luciferase ATP assay.</a> Methods in Molecular Biology. 1105, 589-602 (2014).</li>
</ol>

The authors have nothing to disclose

The bioenergetics extra flux assay serves as an excellent tool to read out the mitochondrial function by measuring oxidative phosphorylation (OXPHOS)-dependent oxygen consumption in real-time. In addition, a glycolytic function which is measured as an extracellular acidification rate (change in extracellular pH) can also be investigated at the same time in real-time analysis.
Successful plating of C. albicans in the assay plate is one of the critical steps in the assay because the inc...

Invasive fungal infections kill over 1.5 million people a year worldwide. This number is on the rise due to an increase in the numbers of people living with compromised immunity, including the elderly, premature infants, transplant recipients, and cancer patients1. C. albicans is an opportunistic human fungal pathogen that is a part of the human microflora. It also inhabits mucosal surfaces and the gastrointestinal tract as a commensal organism. C. albicans produces serious systemic disease in people who have immune deficiencies, who have undergone surgery, or who have been treated with long courses of antibiotics. The Candida species rank among the top three to four causes of nosocomial infectious diseases (NID) in humans2,3,4,5,6,7. The annual global number of Candida bloodstream infections is estimated to be ~400,000 cases, with associated mortalities of 46-75%1. The annual mortality because of candidiasis is roughly 10,000 in the United States alone. The extent of NID caused by fungi is also reflected in astronomical patient expenses5. In the United States, the yearly expense for the treatment of invasive fungal infections surpasses $2 billion, adding a huge strain to already overburdened health care system. Currently, available standard antifungal therapies are limited because of toxicity, increasingly prevalent drug resistance, and drug-drug interactions. Therefore, there is an urgent need to identify new antifungal drug targets that will result in better treatment options for high-risk patients. However, the discovery of new drugs acting on fungal targets is complicated because fungi are eukaryotes. This greatly limits the number of fungal-specific drug targets.
Recent studies have indicated that mitochondria are a critical contributor to the fungal virulence and tolerance to antifungal drugs since mitochondria are important for cellular respiration, oxidative metabolism, signal transduction, and apoptosis8,9,10,11. Both glycolytic and non-glycolytic metabolism are essential for the survival of C. albicans in the mammalian host12,13,14,15,16. Furthermore, several C. albicans mutants lacking mitochondrial proteins, such as Goa1, Srr1, Gem1, Sam37 etc. have been shown to be defective in filamentation, an important virulence factor of C. albicans17,18,19,20,21,22. In addition, these mutants were also shown to be attenuated for virulence in a mouse model of disseminated candidiasis17,18,19,20,21,22. Thus, fungal mitochondria represent an attractive target for drug discovery. However, the study of mitochondrial function in C. albicans is challenging because C. albicans is petite negative23, which means that it cannot survive without the mitochondrial genome.
Here, we describe a protocol that can be used to investigate mitochondrial and glycolytic function in C. albicans without the need to purify mitochondria. This method can also be optimized to investigate the effect of the genetic manipulation or chemical modulators on mitochondrial and glycolytic pathways in C. albicans.

<table><tbody><tr><td>RPMI 1640</td><td>Corning</td><td>MT50020PB</td><td></td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>A8674</td><td></td></tr><tr><td>KCN</td><td></td><td></td><td></td></tr><tr><td>Mito stress kit</td><td>Agilent</td><td>103015-100</td><td></td></tr><tr><td>Oligomycin</td><td>Calbiochem</td><td>495455</td><td></td></tr><tr><td>pH meter</td><td>Accumet</td><td>AR20</td><td></td></tr><tr><td>Phenol red</td><td>Sigma</td><td>P5530</td><td></td></tr><tr><td>Poly-D lysine</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>Rotenone</td><td>Santa cruz</td><td>203242</td><td></td></tr><tr><td>Seahorse XF24 FluxPak</td><td>Agilent</td><td>100850-001</td><td></td></tr><tr><td>SHAM</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Amresco&amp;nbsp;</td><td>241</td><td></td></tr><tr><td>Sodium hydroxie pellets</td><td>J.T Baker</td><td>3722</td><td></td></tr><tr><td>Tissue culture grade water</td><td>Gibco</td><td>1523-0147</td><td></td></tr><tr><td>XF assay calibrant solution</td><td>Agilent</td><td>100840-000</td><td></td></tr><tr><td>Yeast extract Peptone Dextrose</td><td>Fisher scientific,</td><td>BP2469</td><td></td></tr><tr><td>Yeast extract Peptone Dextrose Agar</td><td>Sigma</td><td>A1296</td><td></td></tr><tr><td>Yeast extract Peptone Glycerol</td><td>Sigma</td><td>G2025</td><td></td></tr></tbody></table>

bio-energetics investigation of candida albicans using real-time extracellular flux analysis

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

The focus of this protocol is to determine the bioenergetic functions of C. albicans assessed by extra flux analyzer. A C. albicans mutant lacking mitochondrial protein Mam33 is also included along with its complement strain, mam33Δ/Δ::MAM33 to study the effects of the deletion of a mitochondrial protein on OCR and ECAR. MAM33 encodes for a putative mitochondrial acidic matrix protein and its function in Candida is not known.
<p class="jove...

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

bio-energetics-investigation-candida-albicans-using-real-time

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This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

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

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

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.

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

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

This protocol describes a cost-effective way to perform a glycolysis stress test to quantify metabolic reprogramming in primary mouse microglia. After isolation and inflammatory stimulation, extracellular acidification rate (ECAR) is measured using freshly prepared, pH-adjusted reagents, followed by protein normalization to enable accurate and reproducible assessment of glycolytic shifts.

Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

Learn to measure mitochondrial respiration and glycolytic function in Candida albicans using real-time extracellular flux analysis without mitochondrial purification. This method enables metabolic studies of fungal pathogens.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

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