Assay of absolute magnitude of mitochondrial membrane potential in cells

This protocol describes how to measure the absolute magnitudes and time courses of mitochondrial and plasma membrane potentials in intact cells in adherent cultures. The assay uses fluorescence time-lapse microscopy (epi, confocal or two-photon) to record a time course of tetramethylrhodamine methyl ester (TMRM) and the Molecular Devices FLIPR Plasma Membrane Potential Assay Kit probe (=plasma membrane potential indicator, PMPI) fluorescence. The intact cells are exposed to a calibration paradigm using the reagents described below, and fluorescence responses measured during this challenge are converted into millivolt values using the Membrane Potential Calibration Wizard in Image Analyst MKII.

Equipment

Microscopy requirement:

The assay works with wide field (epifluorescence), confocal and two-photon microscopy.  The epifluorescence microscope has to be capable of low-light level time lapse imaging, e.g. equipped with a fast shutter and a sensitive monochromatic camera. Confocal and two-photon microscopes trivially work in low-light level mode. The image acquisition software must record accurate time stamps for each (pairs of TMRM and PMPI) frames.

Wide field microscopy:

• Inverted microscope with 10x or 20x fluorescence-optimized, high NA (0.5-0.75) lens
• Cooled CCD camera
• Motorized filter turret or filter wheels
• Fast electronic shutter
• Optionally: real time autofocus, such as the Nikon's Perfect Focus System or the Zeiss Definite Focus.

Filter sets: a customized filter set achieves better signal to noise ratio because of less correction required for spectral crossbleed. However fluorescence can be captured by standard green (PMPI) and red (TMRM) emission channels. Ideally TMRM emission is collected above 600nm to decrease crossbleed of PMPI.

Filter	Spectrum (center/bandwidth nm)	Supplier & Cat. #	Note
PMPI excit	500/24	Semrock FF01-500/24-25	Standard YFP cube component
PMPI dichroic	520	Semrock FF520-Di02-25x36	Standard YFP cube component
PMPI emitter	542/27	Semrock FF01-542/27-25	Standard YFP cube component
TMRM excit	580/14	Semrock FF01-580/14-25	individual stock filter
TMRM dichroic	594	Semrock Di02-R594-25x36	individual stock dichroic
TMRM emitter	641/75	Semrock FF02-641/75-25	individual stock filter

Alternatively use the PMPI exciter and dichroic for both TMRM and PMPI channels to record only using an emission filter wheel. Note: the emission bands of PMPI and TMRM are wide, and other similar filters may work just as well.

Confocal microscopy:

• Inverted stage with 10x or 20x fluorescence-optimized, high NA (0.5-0.75) lens
• 488 or 514 nm lines for PMPI excitation
• 543 or 561 lines for TMRM excitation
• Spectral detector set to capture fluorescence similarly to the above specified filters, or filter based detector configured similarly to the above filters

Two-photon microscopy:

• 10x or 20x two-photon microscopy optimized, high NA (0.5-0.75 or 20x NA 1.0 WI) lens
• 930 nm excitation
• Non-descanned detectors with beamsplitter to detect two emission channels simultaneously.
• The above listed emission filters, or alternatively use a 610 nm long pass filter for TMRM.

Small equipment requirement:

• 37C waterbath
• 37C dry or humidified air incubator with no CO2, optionally with a rocker.
• scale, pH meter, pipettes

Reagents and consumables
Reagents stocks:

 

Reagent	Stock Concentration	Solvent	Storage	Supplier & Cat. #	Notes
NaCl	240 mM	H2O	RT	Sigma	sterile filter
KCl	240 mM	H2O	RT	Sigma	sterile filter
KCl	2 M	H2O	RT	Sigma
CaCl2	1 M	H2O	RT	Sigma	sterile filter
Glucose	1 M	H2O	RT	Sigma	sterile filter
Glutamine	200mM (cell culturing)	H2O	-20C	Gibco / Life Technologies
TMRM	50 µM	DMSO	-20C	Life Technologies
PMPI (plasma membrane potential indicator)	(explorer kit vial dissolved in 10ml H2O)	H2O	-20C	Molecular Devices #R8042 FLIPR Membrane Potential Assay Kit (blue) or #R7291 (red)	The bulk kit is 10x more concentrated. Do not filter.
Zosuquidar	25 mM	DMSO	-20C	MedKoo  or Sigma
IAA-94	200 mM	DMSO	-20C	Enzo ALX-550-248-M025	optional component
DIOA	100 mM	DMSO	-20C	Santa Cruz sc-203230A	optional component
Bumetanide	100 mM	DMSO	-20C	Sigma	optional component
TTX (tetrodotoxin)	1 mM	MES/H2O	-20C	Sigma	optional component, dissolve in 10mM MES
Valinomycin	1 mM	Ethanol	-20C	Sigma
Oligomycin	10 mg/ml	Ethanol	-20C	Sigma
Antimycin A	20 mM	Ethanol	-20C	Sigma	Alternatively use myxothiazol
Gramicidin	10 mg/ml	Ethanol	-20C	Sigma
Paraformaldehyde	8%, see below	H2O	-20C		warm up of filter for precipitates
2xPM (potentiometeric medium)	see composition below	H2O	RT or 4C		sterile filter

An abbreviations list is at the end of the document.

2xPM (potentiometric medium)

The 2xPM is a Na-free double concentrated stock solution for composition of the assay medium (potentiometric medium). This is required for a series of mixing steps that ensure constant probe concentrations. The full potentiometric medium is composed in the Assay Protocol section below by mixing the 2xPM with equal amount of NaCl or KCl solutions.

Simple 2xPM:

Substance	mM (2x of final)
KCl	7
MgCl2	2
KH2PO4	0.8
TES	40
NaHCO3	10
Na2SO4	2.4

Set the pH to 7.6 at room temperature using NaOH. Mix an aliquot of 2xPM with 240mM NaCl 1:1, warm up to 37C and adjust the pH with measured amount of HCl or NaOH. Add the required amount to the rest of the 2xPM. Sterile filter and aliquot into 50ml conicals. Store at RT. Note: Don't include CaCl2 into the 2xPM, because calcium phosphate may precipitate.

Cell culture medium-based 2xPM

Custom-order (e.g. from Gibco / Life Technologies) powdered culture medium omitting the following components: NaCl, phenol red, NaHCO3, riboflavin, folic acid, glucose, glutamine and  Ca2+ (in any form). Note: mind the final phosphate concentration in light of the final concentration of added CaCl2!

Substance	mM (2x of final)
Custom culture medium powder (w/o NaCl!)	2x
TES	40 mM
NaHCO3	10 mM

Set the pH to 7.6 at room temperature using NaOH or HCl. Mix an aliquot of 2xPM with 240mM NaCl 1:1, warm up to 37C and adjust the pH with measured amount of HCl or NaOH. Add the required amount to the rest of the 2xPM. Steril filter and aliquot into 50ml conicals. Store at RT. Note: Don't include CaCl2 into the 2xPM, because calcium phosphate may precipitate.

Calibration cocktails:

Anti-swelling cocktail (ASC) - optional

Drug	stock (mM)	Final (uM)	final dilution=	1000	volume=	20
IAA-94	200	100				10
DIOA	100	10				2
Bumetanide	200	80				8
Add DMSO=						0

Mitochondrial depolarization cocktail (MDC)

Drug	stock (mM)	Final (uM)	final dilution=	1000	volume=	100
Valinomycin	1	0.50				50
Oligomycin	10	1				10
Antimycin A	20	1				5
Add EtOH=						35

8% Paraformaldehyde solution:

1. 8g paraformaldehyde for 100 ml distilled water

2. Heat to 60 degrees while stirring. Add drops of 10N NaOH until the solution is transparent (usually 1-4 drops per 100ml sometimes more).

3. Cool to room temperature.

4. Adjust to pH 7.4

5. Filter into aliquot tubes. Store frozen at -20C.

6. Thaw before use in 65C water bath.

Consumables (for working in LabTek coverglass-bottomed chamber or up to 15 wells of a 96-well plate)

• 15 ml conicals 2x
• 1.5 ml  and 2 ml tubes
• Reservoir plate, if using microplate format: polypropylene, 300ul / well volume, round or V bottom, with lid.

Assay protocol:

Generic guidelines for fluorescence microscopic recording of mitochondrial membrane potential:

• Avoid photo-effects: a stable baseline will indicate no photo-toxicity. Use attenuated and shuttered illumination and not too frequent frame captures.
• Acquisition interval: the maximum interval is dictated by how fast will TMRM leak out of the cells after MDC addition - the decay curve must be resolved. In beta-cells 35s cycle time for complete calibration is OK. Higher temporal resolution may exert photo-effects. If using complete (known kt) or relative to baseline calibrations, the acquisition interval can be longer.
• The image acquisition software must record accurate time stamps for each (pairs of TMRM and PMPI) frames.
• The sample needs to be thermostated to 37C, ideally by a heated environment chamber
• Pre-warm all added liquids and pipette tips used for addition
• The sample holder has to be sturdy enough to withstand several liquid additions or removals during the experiment.

Configuration guidelines for Wide field microscopy::

• Exposure time: 50-200ms, balance exposure times to have similar baseline TMRM intensity to end point PMPI intensity
• Illumination intensity: attenuate until image looks grainy - and baseline recordings are stable
• Bin the camera to have ~1-2 um pixels

Configuration guidelines for Confocal microscopy::

• Open pinhole to improve signal intensity
• Laser intensity: attenuate until image looks grainy - and baseline recordings are stable
• Set low image resolution resulting 1-2 um pixels
• Stick to the same gain settings during experiments, keep intensities in the lower part of the detector range, mind that PMPI will brighten during calibration.

Configuration guidelines for Two-photon microscopy :

• Stick to the same gain settings during experiments, keep intensities in the lower part of the detector range, mind that PMPI will brighten during calibration.
• May record z-stacks and set "Mean Intensity" projection in Image Analyst MKII Multi-Dimensional Open dialog Settings tab when opening the recording for analysis.
• Scan fast with frame averaging for better distribution of heat.

Prepare potentiometric medium (PM) working solutions (right before the assay, use it on the same day):

In a 15ml conical prepare supplemented 2xPM with the probes:

Reagent (stock concentration)	amount or concentration in the 2x medium
2xPM	10 ml
CaCl2 (1 M)	1.5 mM (30 ul)
Glucose (1 M)	6 mM (60 ul)
Glutamine (200 mM)	4 mM (200ul)
Zosuquidar (25 mM)	2 uM (0.8ul)
TMRM (50 uM)	10 nM (4ul)
PMPI (explorer kit)	1:200 (100ul)
TPB (1 mM)	1 uM (20 ul)

Mix with vortexing.

1. Save 1ml of this supplemented  2xPM in an 1.5ml tube. Note: a modified culture medium-based 2xPM may form precipitation by time with Ca2+ added. In this case save this 2xPM without Ca2+ added and supplement it before use.

2. Prepare PMNa: in an other 15 ml conical mix 7ml NaCl (240 mM) and 7ml of the above supplemented 2xPM.

3. Prepare PMK: in a 2ml tube mix 1ml KCl (240 mM) and 1ml of the above supplemented 2xPM.

Handling of cultures:

1. Wash cell cultures with PMNa twice. Use 400-500 ul for LabTek 8-well chambered coverglass or 150ul for reduced-area 96-well plate wells.

2. Incubate in a CO2-free warm air incubator, optionally on rocker for 90min

3. Replace medium for fresh PMNa and transfer the culture onto the microscope stage and incubate further 30min. Note: keep the culture warm while transferring to the microscope.

Note: All tubes and reservoirs used for handling PMNa need to be pre-soaked in PMNa, because probes may get depleted by absorption on the walls.

Note: The dilution of 2x media stocks was designed to ensure constant probe concentrations around the sample.

Preparations before time-lapse recording:

While the cell culture dish is preincubating on the microscope stage prepare:

1. Set up acquisition parameters based on the above recommendations (Generic guidelines).

2. Prepare PMNa-MDC by pipetting 500ul PMNa into a pre-soaked tube and add 1.5ul ASC + 1.5 ul MDC + 1.5ul TTX

3. Supplement the PMK tube with 2ul ASC + 2ul MDC + 2ul TTX

4. Mix challenge treatments at 2x concentration with PMNa. If using larger aqueous volumes, e.g. for glucose addition, mix 1M glucose with the supplemented 2xPM at 1:1 resulting a 0.5M glucose stock with identical probe concentration to PMNa. Use this stock to prepare the 2x challenge treatment.

5. Set the volume in the well to support 1:1 additions, e.g. 200ul in a LabTek 8-well chamber or 100ul in a 96-well plate well.

Time-lapse recording:

1. Record a baseline of 30 frames. Longer baselines allow better precision and also a test for non-phototoxic conditions. Stop recording after the baseline. Individual recordings will be merged in Image Analyst MKII.

2. Make the challenge addition by adding the 2x concentrated challenge prepared in PMNa at 1:1, stir well and remove the added volume.

3. Record challenge for an arbitrary length of time

4. Add PMNa-MDC at 2:1 volume to the well and stir well.

5. Quickly start a 20 min recording.

6. In 3-5 steps add (or replace medium) by increasing amounts of supplemented PMK, e.g. 50,100,150, 200 ul for a Labtek 8-well chamber or 10,20,40,75 for reduced are 96-well plate. The volumes are arbitrary, but note down the initial volume and the additions and removals, these values will be required for the calibration. After each addition wait 1 minute, then record 5 frames.

7. Prepare PMK-PFA by mixing 440ul 2xPM 60ul 2M KCl and 500ul 8%PFA supplemented with 1ul of 10mg/ml gramicidin

8. Add PMK-PFA at 1:1

9. Record for another 20-30 min until PMPI fluorescence stabilizes at a maximum level.

The live-cell time lapse experiment ends here. PMK-PFA fixed samples can be processed for immunocytochemistry with any-colored fluorescence labeling as PMPI and TMRM washes out during premeabilization. If the microscope is equipped with appropriate stage motorization, the same view fields can be re-imaged after staining, to pick specific cell types during analysis.

Data analysis in Image Analyst MKII

The analysis of TMRM/PMPI recordings requires spectral unmixing. See related "Calculation of spectral unmixing coefficients for mitochondrial membrane potential measurement" protocol. The spectral unmixing coefficient matrix qualifies the configuration of the microscope, so it has to be re-measured if the relevant microscope configuration changes, but not for different specimens. The ratio of the two (TMRM and PMPI) exposure times affects the coefficients, but a change exposure time can be accounted for by changing exposure correction parameters in the Spectral Unmixing function by editing the pipeline.

Analysis of the recording:

1. Open the recording using the File/”Open Image Series/Measurement” or the  toolbar button. In the Open Image/Measurement dialog select all recorded files comprising the time lapse.  Alternatively use multiple file selection in the Windows Explorer and drag-and-drop on the Image Analyst MKII. Note: check the correct order in the Files tab of the Multi-Dimensional Open dialog.

2. Select the “Mitochondrial membrane potential measurement (TMRM/PMPI)” pipeline in the Pipelines/”Intensity Measurements”/Applications main menu point.

3. Configure the pipeline as follows:

   1. Set channel number associations for TMRM and PMPI

   2. Use a smaller background level (10-30 percentile) for confluent cultures and ~50 percentile for sparse cultures. Percentiles below 5-10 may increase noise.

   3. Enter the coefficient matrix as above calculated.

   4. Set cell diameter in pixels. To measure it open the recording, draw linear ROI across a typical cell using the   toolbar, and then reading out its length by double-clicking the status bar of the image to bring up an Image Property window. (Alternatively use the context menu of the Image Window).

   5. If you have previously saved the calibration configuration, enter the file file name into the corresponding pipeline parameter or leave this field blank.

4. Press the  button on the main toolbar or in the bottom of the Multi-Dimensional Open dialog.

5. To calibrate PMPI and fluorescence traces to millivolts, either follow the step-by-step configuration of the “Membrane Potential Calibration Wizard” (below) or open the “calibration configuration.ips” configuration file accompanying the image data files.

6. Press the  button to perform the calibration or the  button to transfer calibrated potentials to Excel.

Configuration of the Membrane Potential Calibration Wizard:

We used this technology also in the following paper:

3. Birket MJ, Orr AL, Gerencser AA, Madden DT, Vitelli C, Swistowski A, Brand MD, and Zeng X. A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells. Journal of Cell Science, 2011 24:348-58.