Fluorescence‐Based Microplate Assays for In Vitro Assessment of Mitochondrial Toxicity, Metabolic Perturbation, and Cellular Oxygenation

James Hynes1, Conn Carey1, Yvonne Will2

1 Luxcel Biosciences, BioInnovation Centre, University College Cork, Cork, 2 Pfizer Global Research, Groton, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.16
DOI:  10.1002/cptx.3
Online Posting Date:  November, 2016
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Abstract

High‐throughput in vitro cell metabolism assays are of particular use for identification and delineation of mitochondrial toxicity and related metabolic perturbation. Here, a panel of fluorescence‐based metabolism assays are described for measuring oxygen consumption, glycolytic flux, and cellular oxygenation. They can be applied to analysis of both isolated mitochondria and cell models. Sample data are presented illustrating how these protocols can be used to examine drug treatment, the interplay between oxidative and glycolytic ATP generation, and the impact of cell oxygenation on this balance. Descriptions are provided on how these measurements can be applied to 3D systems and how they can be multiplexed with other relevant metabolic readouts. Mitochondrial isolation and cell permeabilization protocols are also provided. © 2016 by John Wiley & Sons, Inc.

Keywords: 3D culture; cellular oxygenation; glycolysis; mitochondria; oxygen consumption; toxicity

     
 
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Table of Contents

  • Introduction
  • Measurement of Oxygen Consumption in Isolated Mitochondria
  • Basic Protocol 1: Measurement of Electron Transport Chain Activity
  • Support Protocol 1: Isolation of Mitochondria from Rat Liver
  • Support Protocol 2: Mitochondrial Protein Assay
  • Support Protocol 3: Instrument Signal Check
  • Measurement of Metabolic Perturbation in Whole Cells
  • Basic Protocol 2: Cell‐Based Measurement of Oxygen Consumption
  • Support Protocol 4: Cell Permeabilization
  • Basic Protocol 3: Cell‐Based Measurement of Glycolytic Flux
  • Measuring Cellular Oxygenation and Associated Metabolic Shifts
  • Basic Protocol 4: Measurement of Cellular Oxygenation Using Fluorescent Intracellular Oxygen Probes
  • Support Protocol 5: Oxygen Calibration of the Fluorescence Plate Reader Using MitoXpress‐Intra
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Electron Transport Chain Activity

  Materials
  • MitoXpress‐Xtra HS Oxygen Consumption Assay kit (Luxcel Biosciences, cat. no. MX‐200), including high‐sensitivity (HS) mineral oil
  • Measurement buffer 1 (see recipe)
  • Substrate stock solution (select one; store in aliquots at −20°C):
  • 1 M sodium succinate in water, pH 7.4
  • 0.5 M glutamate/0.5 M malate in water, pH 7.4
  • 100 mM adenosine 5′‐diphosphate (ADP) in water (store in aliquots at −20°C)
  • Isolated mitochondria (see protocol 2)
  • Test compounds in appropriate solvent
  • Positive control compounds: rotenone and p‐trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP)
  • Fluorescence plate reader (Table 2.16.1) with temperature control and kinetic analysis software
  • 30°C water bath
  • 96‐well plates, black with clear bottom
  • Multiblock plate heater (VWR, cat. no. 12621‐108)
  • Multichannel pipetter
  • Syringe dispenser (Eppendorf) with 2.5‐ml plastic syringes
Table 2.6.1   MaterialsRecommended Settings for Common Fluorescence Microplate Readers a

Manufacturer Instrument Optical configuration Reads b Optimum mode Excitation/emission wavelengths (nm)
For MitoXpress measurements:
BMG Labtech FLUOstar Omega/POLARstar Omega
  • Filter‐based
  • Top or bottom read
  • 30/30 µsec
  • 70/30 µsec
Dual‐read TR‐F (lifetime)
  • 340 ± 50 nm (TR‐EX L)
  • 655 ± 50 nm (BP‐655)
CLARIOstar
  • Filter‐based
  • Bottom read
  • 30/30 µsec
  • 70/30 µsec
Dual‐read TR‐F (lifetime)
  • 340 ± 50 nm (TR‐EX)
  • 665 ± 50 nm or 645 ± 20 nm with LP‐TR dichroic
PHERAstar FS
  • Filter‐based
  • Top or bottom read
  • 40/100 µsec
  • N/A
TR‐F
  • 337 nm (HTRF module)
  • 665 nm (HTRF Module)
FLUOstar Optima/POLARstar Optima
  • Filter‐based
  • Top or bottom read
  • 30/100 µsec
  • N/A
TR‐F
  • 340 ± 50 nm(TR‐EX L)
  • 655 ± 50 nm (BP‐655)
Perkin Elmer VICTOR X4/X5
  • Filter‐based
  • Top read
  • 30/30 µsec
  • 70/30 µsec
Dual‐read TR‐F (lifetime)
  • 340 ± 40 nm (D340)
  • 642 ± 10 nm (D642)
EnVision
  • Filter‐based
  • Top read
  • 40/100 µsec
  • N/A
TR‐F
  • 340 nm ± 60 nm (X340)
  • 650 nm ± 8 nm (M650)
BioTek Cytation 3/5
  • Filter‐based
  • Top or bottom read
  • 30/30 µsec
  • 70/30 µsec
Dual‐read TR‐F (lifetime)
  • 380 ± 20 nm
  • 645 ± 15 nm
Synergy H1/Neo2
  • Filter‐based
  • Top or bottom read
  • 30/30 µsec
  • 70/30 µsec
Dual‐read TR‐F (lifetime)
  • 380 ± 20 nm
  • 645 ± 15 nm
Tecan Spark 10M
  • Filter‐based
  • Top or bottom read
  • 30/30 µsec
  • 70/30 µsec
Dual‐read TR‐F (lifetime)
  • 380 ± 20 nm
  • 650 ± 20 or 670 ± 40 nm
Infinite M1000 Pro/F200 Pro
  • Monochromator/filter
  • Top or bottom read
  • 30/30 µsec
  • 70/30 µsec
Dual‐read TR‐F (lifetime)
  • 380 ± 20 nm
  • 650 ± 20 or 670 ± 40 nm
Infinite M200 Pro/Safire/Genios Pro
  • Monochromator/filter
  • Top or bottom read
  • 30/100 µsec
  • N/A
TR‐F
  • 380 ± 20 nm
  • 650 ± 20 nm
Molecular Devices Gemini/Flexstation
  • Monochromator
  • Top or bottom read
  • N/A
  • N/A
Intensity (Prompt)
  • 380 nm
  • 650 nm
For pH‐Xtra measurements:
BMG Labtech FLUOstar Omega/POLARstar Omega
  • Filter‐based
  • Top or bottom read
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 340 ± 50 nm (TR‐EX L)
  • 615 ± 10 nm (BP‐615)
CLARIOstar
  • Filter‐based
  • Top or bottom read
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 340 ± 50 nm (TR‐EX L)
  • 615 ± 10 nm (BP‐615)
PHERAstar FS
  • Filter‐based
  • Top read (HTRF module)
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 337 nm
  • 620 nm
FLUOstar Optima/POLARstar Optima
  • Filter‐based
  • Top or bottom read
  • 100/100 µsec
  • N/A
TR‐F
  • 340 ± 50 nm(TR‐EX L)
  • 615 ± 10 nm (BP‐615)
Perkin Elmer VICTOR X4/X5
  • Filter‐based
  • Top read
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 340 ± 40 nm (D340)
  • 615 ± 8.5 nm (D615)
EnVision
  • Filter‐based
  • Top read
  • 100/50 µsec
  • 300/50 µsec
Dual‐read TR‐F (lifetime)
  • 340 nm ± 60 nm (X340)
  • 615 nm ± 8.5 nm (M615)
BioTek Cytation 3/5
  • Filter‐based
  • Top or bottom read
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 360 ± 40 nm
  • 620 ± 10 nm
Synergy H1/Neo2
  • Filter‐based
  • Top or bottom read
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 360 ± 40 nm
  • 620 ± 10 nm
Tecan Spark 10M
  • Monochromator‐based
  • Top or bottom read
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 380 ± 20 nm
  • 615 ± 10 nm
Infinite M1000 Pro/F200 Pro
  • Monochromator/filter
  • Top or bottom read
  • 100/30 µsec
  • 300/30 µsec
Dual‐read TR‐F (lifetime)
  • 380 ± 20 nm
  • 615 ± 10 nm
Infinite M200 Pro/Safire/Genios Pro
  • Monochromator/filter
  • Top or bottom read
  • 100/100 µsec
  • N/A
TR‐F
  • 380 ± 20 nm
  • 615 ± 10 nm

 aWhere available, filter‐based dual‐read (lifetime) TR‐F is recommended, particularly for pH‐Xtra and MitoXpress‐Intra measurements. It is required for quantitative analyses, including data transposition into H+ and O 2 scales.
 bThe upper number represents Read 1 (D1/W1) and the lower number represents Read 2 (D2/W2).

Support Protocol 1: Isolation of Mitochondria from Rat Liver

  Materials
  • Male Sprague‐Dawley rats (150 to 180 g; e.g., Charles River)
  • Carbon dioxide source
  • Isolation buffers 1 and 2 (see reciperecipes), ice cold
  • Sterile dissection tools
  • 100‐ml glass tissue homogenizer with Teflon pestle
  • Power drill (hand‐held or static)
  • Cheesecloth (VWR cat. no. 21910‐105)
  • Glass stirring rods

Support Protocol 2: Mitochondrial Protein Assay

  Materials
  • 2 mg/ml bovine serum albumin (BSA)
  • 1% (v/v) Triton X‐100
  • Mitochondrial sample (see protocol 2)
  • BCA kit (Pierce) containing protein reagents A and B
  • 2‐ml microcentrifuge tubes
  • Standard 96‐well plate
  • 37°C heating block or incubator
  • Absorbance plate reader

Support Protocol 3: Instrument Signal Check

  Materials
  • Probe kit (select one):
  • MitoXpress‐Xtra kit, including high‐sensitivity (HS) mineral oil
  • pH‐Xtra kit, including respiration buffer tablet
  • Probe diluent: water, PBS, or culture medium
  • Culture medium
  • Time‐resolved fluorescence plate reader with temperature control and kinetic analysis software
  • 96‐well plate, black with clear bottom
  • Multiblock heater (VWR, cat. no. 12621‐108)
  • Syringe dispenser (Eppendorf) with 2.5‐ml plastic syringes

Basic Protocol 2: Cell‐Based Measurement of Oxygen Consumption

  Materials
  • Cells to be tested
  • Growth medium supplemented for normal growth
  • MitoXpress‐Xtra HS Oxygen Consumption Assay kit (Luxcel Biosciences, cat. no. MX‐200), including high‐sensitivity (HS) mineral oil
  • Test compounds and appropriate solvents
  • 96‐well plates, black with clear bottom
  • Standard 37°C, 5% CO 2, humidified incubator
  • Time‐resolved fluorescence plate reader (Table 2.16.1) with temperature control and kinetic analysis software
  • 37°C water bath
  • 50‐ml plastic tube
  • Aspirator or microplate centrifuge
  • Multichannel or repeat pipetter
  • Multiblock heater (VWR, cat. no. 12621‐108)
  • Syringe dispenser (Eppendorf) with 2.5‐ml plastic syringes

Support Protocol 4: Cell Permeabilization

  Additional Materials (also see protocol 5)
  • Measurement buffer 2 (see recipe)
  • 20 mM ADP
  • 3 mg/ml digitonin
  • 75 µM rotenone (150×)
  • 150 μM antimycin A (150×)
  • 100 mM succinate (10×)

Basic Protocol 3: Cell‐Based Measurement of Glycolytic Flux

  Materials
  • Cells to be tested
  • Growth medium supplemented for normal growth
  • pH‐Xtra Glycolysis Assay kit (Luxcel Biosciences, cat. no. PH‐200), including respiration buffer tablet
  • Test compounds and appropriate solvents
  • 96‐well plates, black with clear bottom
  • Standard 37°C, 5% CO 2, humidified incubator
  • CO 2‐free, 37°C, 95% humidified incubator
  • pH meter
  • Time‐resolved fluorescence plate reader (Table 2.16.1) with temperature control and kinetic analysis software
  • 0.22‐µm syringe filter
  • 37°C water bath
  • Aspirator or microplate centrifuge
  • Multichannel or repeat pipetter
  • Multiblock heater (VWR, cat. no. 12621‐108)

Basic Protocol 4: Measurement of Cellular Oxygenation Using Fluorescent Intracellular Oxygen Probes

  Materials
  • Cells to be tested
  • Culture medium
  • MitoXpress‐Intra (Luxcel Biosciences, cat. no. MX‐300)
  • Measurement buffer 4 (see recipe)
  • Test compounds and appropriate solvents
  • Measurement buffer 1 (see recipe)
  • 96‐well plate, black with clear bottom
  • Standard CO 2 incubator
  • CO 2‐free, 37°C, 95% humidified incubator
  • 37°C water bath
  • Multiblock heater (VWR, cat. no. 12621‐108)
  • Aspirator
  • Multichannel or repeat pipetter
  • Appropriate time‐resolved fluorescence plate reader (Table 2.16.1)
  • 50‐ml plastic tube

Support Protocol 5: Oxygen Calibration of the Fluorescence Plate Reader Using MitoXpress‐Intra

  Additional Materials (also see protocol 8)
  • 150 μM antimycin A
  • 1 mg/ml glucose oxidase (GOx)
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Figures

Videos

Literature Cited

Literature Cited
  Chapple, S.J., Keeley, T.P., Mastronicola, D., Arno, M., Vizcay‐Barrena, G., Fleck, R., Siow, R.C.M., and Mann, G.E. 2016. Bach1 differentially regulates distinct Nrf2‐dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels. Free Radic. Biol. Med. 92:152‐162. doi: 10.1016/j.freeradbiomed.2015.12.013.
  Hynes, J., Marroquin, L.D., Ogurtsov, V.I., Christiansen, K.N., Stevens, G.J., Papkovsky, D.B., and Will, Y. 2006. Investigation of drug‐induced mitochondrial toxicity using fluorescence‐based oxygen‐sensitive probes. Toxicol. Sci. Off. J. Soc. Toxicol. 92:186‐200. doi: 10.1093/toxsci/kfj208.
  Hynes, J., O'Riordan, T.C., Zhdanov, A.V., Uray, G., Will, Y., and Papkovsky, D.B. 2009. In vitro analysis of cell metabolism using a long‐decay pH‐sensitive lanthanide probe and extracellular acidification assay. Anal. Biochem. 390:21‐28. doi: 10.1016/j.ab.2009.04.016.
  Hynes, J., Nadanaciva, S., Swiss, R., Carey, C., Kirwan, S., and Will, Y. 2013. A high‐throughput dual parameter assay for assessing drug‐induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays. Toxicol. In Vitro 27:560‐569. doi: 10.1016/j.tiv.2012.11.002.
  Lapidus, R.G. and Sokolove, P.M. 1993. Spermine inhibition of the permeability transition of isolated rat liver mitochondria: An investigation of mechanism. Arch. Biochem. Biophys. 306:246‐253. doi: 10.1006/abbi.1993.1507.
  Marroquin, L.D., Hynes, J., Dykens, J.A., Jamieson, J.D., and Will, Y. 2007. Circumventing the Crabtree effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97:539‐547. doi: 10.1093/toxsci/kfm052.
  Nadanaciva, S. and Will, Y. 2009. Current concepts in drug‐induced mitochondrial toxicity. Curr. Protoc. Toxicol. 40:2.15.1‐2.15.9. doi: 10.1002/0471140856.tx0215s40.
  National Research Council of the National Academies (NRC). 2011. Guide for the Care and Use of Laboratory Animals, 8th ed. The National Academies Press, Washington, D.C.
  Ogurtsov, V.I., Hynes, J., Will, Y., and Papkovsky, D.B. 2008. Data analysis algorithm for high throughput enzymatic oxygen consumption assays based on quenched‐fluorescence detection. Sens. Actuators B Chem. 129:581‐590. doi: 10.1016/j.snb.2007.09.004.
  O'Riordan, T.C., Fitzgerald, K., Ponomarev, G.V., Mackrill, J., Hynes, J., Taylor, C., and Papkovsky, D.B. 2007. Sensing intracellular oxygen using near‐infrared phosphorescent probes and live‐cell fluorescence imaging. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292:R1613‐R1620. doi: 10.1152/ajpregu.00707.2006.
  Sung, H.J., Ma, W., Wang, P.Y, Hynes, J., O'Riordan, T.C., Combs, C.A., McCoy, J.P. Jr., Bunz, F., Kang, J.G., and Hwang, P.M. 2010. Mitochondrial respiration protects against oxygen‐associated DNA damage. Nat. Commun. 1:5. doi: 10.1038/ncomms1003.
  Toussaint, O., Weemaels, G., Debacq‐Chainiaux, F., Scharffetter‐Kochanek, K., and Wlaschek, M. 2011. Artefactual effects of oxygen on cell culture models of cellular senescence and stem cell biology. J. Cell. Physiol. 226:315‐321. doi: 10.1002/jcp.22416.
  Wallace, K.B. 2008. Mitochondrial off targets of drug therapy. Trends Pharmacol. Sci. 29:361‐366. doi: 10.1016/j.tips.2008.04.001.
  Will, Y., Hynes, J., Ogurtsov, V.I., and Papkovsky, D.B. 2007. Analysis of mitochondrial function using phosphorescent oxygen‐sensitive probes. Nat. Protoc. 1:2563‐2572. doi: 10.1038/nprot.2006.351.
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