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Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime
利用NAD(P)H荧光强度和寿命评估细胞氧化还原状态   

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Abstract

NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states.

Keywords: NADH(NADH), NADPH(NADPH), NAD(P)H(NAD(P)H), Autofluorescence(自体荧光), Microscopy(显微镜检查), Fluorescence lifetime(荧光寿命), FLIM(FLIM), Redox state(氧化还原状态)

Background

The reduced form of the redox cofactor nicotinamide adenine dinucleotide (NADH) and its phosphorylated counterpart NADPH are intrinsically fluorescent, both absorbing light at wavelengths of 340 (± 30) nm and emitting at 460 (± 50) nm (Patterson et al., 2000). These spectral characteristics are lost upon oxidation to NAD+ or NADP+ (De Ruyck et al., 2007). The redox balances of the separate NAD and NADP pools dictate contrasting metabolic processes (Ying, 2008), as shown in Figure 1. NAD acts as an electron acceptor for the oxidation of sugar, lipid and amino acid substrates in the mitochondria by the tricarboxylic acid (TCA) cycle and as an electron donor to the electron transport chain (ETC) on the inner mitochondrial membrane (IMM), fuelling the pumping of protons into the intermembrane space to act as a power source for the synthesis of adenosine triphosphate (ATP) by the F1FO ATP synthase (Osellame et al., 2012). The balance of NADH to NAD+ in the mitochondria therefore reflects the balance of TCA cycle to ETC activity. ETC dysfunction causes increases in the NADH/NAD+ ratio and the production of potentially damaging reactive oxygen species (ROS) (Murphy, 2009). The cell’s antioxidant defences require the NADP pool to provide reducing equivalents for their maintenance, so the NADPH/NADP+ ratio must be maintained high (> 3) (Pollak et al., 2007). The redox state of the mitochondrial NAD pool and the relative abundance of NADPH are therefore key factors in the level of oxidative stress in a cell type.


Figure 1. Schematic outline of mitochondrial NAD(P)H metabolism. Substrate oxidation in the TCA cycle passes electrons to NAD+, forming NADH. A. Under resting conditions, electrons carried by NADH are passed along the ETC, powering the pumping of protons from the mitochondrial matrix across the inner mitochondrial membrane (IMM) into the intermembrane space. The resulting proton gradient powers the production of ATP at complex V of the ETC (F1FO ATP synthase). B. Addition of an uncoupler such as carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) allows protons to leak back across the IMM, causing the rate of oxidation of NADH at the ETC to increase to restore the membrane gradient. C. Inhibition of the ETC by rotenone halts NADH oxidation and causes an increase in the production of superoxide (O2-), the proximal source of mitochondrial ROS. This is neutralised upon its conversion into water by the superoxide dismutase (SOD2) and glutathione (GSH/GSSG) antioxidant defence systems, maintained by NADPH.

Here, we describe protocols for the assessment of the mitochondrial NADH/NAD+ ratio and the NADPH/NADH balance that rely on the fluorescence of these cofactors when reduced. Their identical absorption and emission spectra leads the combined signal to be termed NAD(P)H (Blacker and Duchen, 2016). Measuring the change in NAD(P)H fluorescence using a confocal microscope following the application of an ETC uncoupler and inhibitor allows the mitochondrial NADH/NAD+ balance to be estimated (Duchen et al., 2003). To discriminate between the relative contributions of NADH and NADPH to the total signal, fluorescence lifetime imaging microscopy (FLIM) must be introduced (Blacker et al., 2014). These protocols describe, in further detail, methods used in Tosatto et al. (2016) to investigate the role of the selective channel responsible for mitochondrial calcium uptake, the mitochondrial calcium uniporter (MCU), in the progression of breast cancer. Basic understanding of confocal microscopy is assumed. For background, readers are directed to Pawley et al. (2012).

Materials and Reagents

  1. NuncTM cell culture treated EasYFlasksTM (75 cm2, filter closure) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156499 )
  2. Falcon tubes (15 ml) (VWR, catalog number: 734-0451 )
  3. Tips  
  4. Falcon tubes (50 ml) (VWR, catalog number: 734-0448 )
  5. Eppendorf tubes (1.5 ml) (VWR, catalog number: 211-2520 )
  6. NuncTM cell-culture treated multidishes (6 wells) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  7. Coverslips (22 mm, thickness No.1) (VWR, catalog number: 631-0158 )
  8. Syringe (20 ml, VWR, catalog number: 613-3921 )
  9. Filter steriliser (0.2 μm, VWR, catalog number: 514-0064 )
  10. MDA-MB-231 cells (ATCC, catalog number: HTB-26 )
  11. Advanced Dulbecco’s modified Eagle’s medium (DMEM) (500 ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 12491023 )
  12. Fetal bovine serum (FBS, heat inactivated, 50 ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
  13. GlutaMAXTM (5 ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  14. Penicillin-streptomycin (10,000 U ml-1, 5 ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  15. Trypsin-EDTA (0.25%, with phenol red, 100 ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  16. FCCP (10 mg) (Sigma-Aldrich, catalog number: C2920 )
  17. Rotenone (5 g) (Sigma-Aldrich, catalog number: R8875 )
  18. Ethanol (1 L) (Sigma-Aldrich, catalog number: 02860 )
  19. Dulbecco’s modified Eagle’s medium powder (without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate) (Sigma-Aldrich, catalog number: D5030 )
  20. D-(+) glucose (100 g) (Sigma-Aldrich, catalog number: G8270 )
  21. Sodium pyruvate (25 g) (Sigma-Aldrich, catalog number: P2256 )
  22. HEPES (100 g) (Sigma-Aldrich, catalog number: H3375 )
  23. Sodium hydroxide solution (1 N, 1 L) (Sigma-Aldrich, catalog number: 71463 )
  24. Hydrochloric acid solution (1 N, 1 L) (Sigma-Aldrich, catalog number: 71763 )
  25. Routine cell culture medium (500 ml) (see Recipes)
  26. Live-cell imaging medium (50 ml) (see Recipes)
  27. ETC perturbations (see Recipes)

Equipment

  1. Incubator (37 °C, 5% CO2) (Thermo Fisher Scientific, Thermo ScientificTM, model: HERAcellTM 150i )
  2. Centrifuge with 15 ml Falcon tube capacity (Mega Star 1.6) (VWR, catalog number: 521-1749 )
  3. Haemocytometer (Neubauer) (VWR, catalog number: 630-1509 )
  4. Laser-scanning microscope (Zeiss, model: LSM510 )
    1. 40x magnification objective
    2. Blackout curtains, for room and covering FLIM microscope (see Figure 2)



      Figure 2. Equipment for high signal to noise FLIM. A. Zeiss LSM510 with Coherent Chameleon for two-photon excitation and Becker & Hickl HPM-100-40 detector and SPC830 counting electronics. B. Blackout curtains surrounding both the microscope room and the microscope itself act to ensure that background noise in the FLIM images is kept to a minimum.

    3. Emission filters for NAD(P)H fluorescence (435-485 nm)
    4. For single-photon excitation:
      1. 351 nm laser source (Enterprise UV, Coherent)
      2. Long-pass dichroic (375 nm cutoff)
      3. Quartz microscope optics
    5. For two-photon excitation:
      1. Ti:sapphire laser modelocked at 720 nm (Chameleon Ultra, Coherent)
      2. Short pass dichroic (650 nm cutoff)
      3. Non-descanned detection
    6. For FLIM:
      1. Detector with single photon sensitivity (Becker & Hickl, model: HPM-100-40)
      2. Time-correlated single photon counting (TCSPC) electronics (Becker & Hickl, model: SPC-830)
      3. Pulsed excitation source (two-photon excitation by Ti:sapphire laser at 720 nm; Chameleon Ultra, Coherent)
    7. Heated microscope stage (custom built to hold imaging rings, see Figure 3)



      Figure 3. Apparatus for mounting coverslips on the microscope. A. Custom built rings and mounting block for securing 22 mm circular coverslips. B. Coverslips are placed directly onto the base of a metal ring. C. A concentric ring with a rubber seal is placed on top of the coverslip. D. A third ring is screwed onto the first two in order to form a watertight seal over the coverslip, allowing (E) the imaging buffer to be pipetted onto the cells. F. The imaging ring and cells then fit into a heated microscope stage.

  5. Straight-tip forceps (VWR, catalog number: 232-0094 )
  6. Imaging rings (custom built, see Figure 3)
  7. Inverted, phase-contrast, bench top microscope (VWR, catalog number: 630-2145 ; ZEISS, model: Primovert )
  8. Laboratory balance (0.1 mg resolution) (Mettler-Toledo, catalog number: 30029067 )
  9. pH meter (Mettler-Toledo International, catalog number: 30266626 )
  10. Tally counter (VWR, catalog number: 720-1984 )

Software

  1. Microscope control software (Zeiss ZEN)
  2. FLIM acquisition software (Becker & Hickl SPCM)
  3. FLIM fitting software (Becker & Hickl SPCImage)
  4. Image analysis software (NIH ImageJ)

Procedure

  1. Routine culture of MDA-MB-231 cells
    1. Culture control (scrambled plasmid) and MCU knockdown MDA-MB-231 cells in Advanced DMEM supplemented with 10% FBS, 2 mM GlutaMAXTM, 100 U ml-1 penicillin and 100 μg ml-1 streptomycin.
    2. Maintain cells as monolayers in 75 cm2 tissue culture flasks containing 10-12 ml growth medium in a 37 °C, 5% CO2 incubator.
    3. Passage every 3-4 days is ensured by splitting at a 1:10 ratio at 70-80% confluence.
    4. Dissociate cells by an initial wash with 2 ml trypsin followed by incubation with 2 ml fresh trypsin for 5 min.

  2. Plating of cells for microscopy
    1. Discard growth media from 75 cm2 flask and detach cells by incubating with 3 ml trypsin for 5 min.
    2. Collect cells and inhibit the enzyme by adding 5 ml growth medium.
    3. Transfer the contents of the flask to a 15 ml Falcon tube and centrifuge at 400 x g for 5 min at room temperature (24 °C).
    4. Resuspend cells in 5 ml fresh medium and transfer a small amount (~100 μl) to an Eppendorf tube for cell counting.
    5. Count cell concentration in resuspension using haemocytometer and tally counter on bench top microscope, ensuring to mix contents of the Eppendorf tube thoroughly with pipette to account for settling.
    6. Using results of cell counting, pipette 1,800,000 cells from cell suspension into a new 50 ml Falcon tube (300,000 cells per well of a 6 well plate). Top up tube to contain a total of 12 ml (2 ml total medium per well).
    7. Using forceps, transfer one coverslip into each well of a 6 well plate.
    8. Pipette 2 ml of the 1,800,000 cell mix into each well, again ensuring to mix contents of the tube using pipette prior to transfer.
    9. Place 6 well plate in incubator and leave overnight to allow attachment of cells to coverslips prior to imaging the next day.
    10. Repeat process for each cell type under analysis in a separate 6 well plate.

  3. NAD redox state assay
    1. Use forceps to transfer a coverslip from the 6 well plate into the metal imaging ring (see Figure 3). Tighten until a small amount of resistance is felt to ensure a watertight seal.
    2. Wash cells by gently (to ensure cells are not detached) pipetting 400 μl of imaging buffer onto coverslip in order to remove remaining phenol red.
    3. Dispose washing medium and gently pipette 400 μl of imaging buffer onto cells.
    4. Transfer coverslip onto heated stage of confocal microscope and bring cells into focus under brightfield illumination.
    5. With 375 nm long pass dichroic and 435-485 nm emission filters in place, gradually increase intensity of 351 nm excitation laser until NAD(P)H fluorescence is clearly observed.
    6. Search coverslip and magnify image such that 10-20 cells can be observed in the field of view.
    7. Begin a time-series acquisition, imaging the field of view every 2 min.
    8. After 4 images (6 min from the start of time-series), gently add 100 μl of 5 μM FCCP solution directly onto cells, giving a final FCCP concentration of 1 μM. The NAD(P)H fluorescence signal should begin to decrease due to increased ETC activity.
    9. Following acquisition of 4 images under FCCP treatment (8 min after addition), gently add 100 μl of 30 μM rotenone solution directly onto cells, giving a final rotenone concentration of 5 μM. The NAD(P)H fluorescence signal should begin to increase, due to inhibition of the ETC.
    10. Terminate experiment 10 min after rotenone addition, giving a total experiment time of 24 min.
    11. Dispose of cells and use 100% ethanol to clean any experimental equipment that may come into contact with FCCP or rotenone (e.g., imaging rings, microscope objective).
    12. Repeat procedure for all coverslips and save data for subsequent analysis.

  4. NAD(P)H FLIM assay
    1. Prepare a coverslip in the metal imaging ring with 400 μl imaging buffer (identically as for the redox state assay) and mount on microscope, adjusting focus to observe cells clearly under brightfield illumination.
    2. With 650 nm short pass dichroic and 435-485 nm emission filters in place, gradually increase intensity of 720 nm excitation laser until NAD(P)H fluorescence can be observed with the internal detectors of the microscope.
    3. Search coverslip and magnify image such that 10-20 cells can be observed in the field of view.
    4. Adjust microscope settings to send fluorescence signal to non-descanned port where FLIM detector is located.
    5. Begin FLIM data acquisition, scanning for 2 min total.
    6. Move to a new location on the coverslip and repeat process. Acquire 3-5 images per coverslip.
    7. Dispose of cells and use 100% ethanol to clean any experimental equipment that may come into contact with FCCP or rotenone (e.g., imaging rings, microscope objective).
    8. Repeat procedure for all coverslips and cell types.

Data analysis

  1. NAD redox state assay (see Figure 4)


    Figure 4. Schematic representation of the mitochondrial NAD redox state assay. The fluorescence intensities following treatment with 1 μM FCCP and 1 μM rotenone are used as the dynamic range through which the resting fluorescence intensity, and thereby the resting percentage of reduced NADH, can be assessed. Thresholding is used to select pixels above a defined brightness value (i.e., pixels containing cells), reducing the impact of background noise.

    1. Import time series images into ImageJ. Dataset will be represented as a ‘stack’, with the x- and y-axes representing the spatial coordinates of the images and the z-axis representing the time since the start of the experiment. The time series can be navigated by moving the horizontal scroll bar.
    2. Use the threshold tool to highlight cell regions in red. The minimum and maximum values should be chosen such that no background signal is selected at any of the time points, and no bright intracellular regions (e.g., with rotenone) should be ignored.
    3. Ensure that measurements are limited to thresholded regions, negating the impact of background fluorescence, by clicking ‘Limit to threshold’ in the ‘Set Measurements’ dialog.
    4. Plot the time evolution of the NAD(P)H fluorescence signal by selecting Plot Z-axis Profile from the Stacks menu. Export the data to Microsoft Excel.
    5. Repeat this process for each time-series acquired.
    6. In Excel, scale the intensities at each time point F(t) as a percentage of the minimum (with FCCP) and maximum (with rotenone) values of its time-series by applying the following formula:

    7. Correct application of this procedure will leave the minimum value of each time-series at 0% and the maximum value as 100%.
    8. For each cell line, take a mean of all scaled data points acquired under resting conditions to calculate the final Reduced NADH (%) value. Express the uncertainty as the standard error of these points. Statistical significance of any differences between the Reduced NADH (%) values for each cell line can be assessed using a Wilcoxon signed-rank test.
  2. NAD(P)H FLIM assay (see Figure 5)


    Figure 5. Software for FLIM analysis. Fitting of biexponential fluorescence decays to a FLIM image is performed in SPCImage (A). Parameter matrices are exported to ImageJ (B) for subsequent analysis. Regions of interest containing cytosol and mitochondria are created by combining a mask of the entire cell, generated by thresholding, and excluding the nuclear regions, drawn by hand.

    1. In SPCImage, import the FLIM image and adjust the settings to fit 2 decay components at each pixel.
    2. To ensure fits of sufficient accuracy, binning should be increased until more than 100 counts are contained in the peak of the decay in the dimmest cytosolic pixel of interest (Blacker et al., 2014). For 2 min acquisitions with laser powers kept sufficiently low to avoid cell damage, this typically requires a binning factor of 2 to 4, corresponding to the summation of data from between 24 and 80 surrounding pixels.
    3. Begin the fitting procedure at each pixel by choosing Calculate Decay Matrix. This process could take up to 5 min, depending on the power of the computer being used.
    4. Export matrices of each decay parameter - α2 (%), ,  - and the photon counts at each pixel for subsequent analysis in ImageJ.
    5. Import α2 (%), ,  and photon count images into ImageJ using the Import Text Image command.
    6. Use thresholding and the Create Selection command on the photon count image to add a region of interest (ROI) containing the cells to the ROI manager.
    7. Use the polygon selection tool to create a ROI of the cell nuclei and save to ROI manager. The nuclei typically have altered NAD(P)H fluorescence decay characteristics, due to differences in metabolism to the rest of the cell, so are neglected from the analysis here for simplicity.
    8. Create a mask from the cell ROI. Select the nuclear ROI when in the mask window and fill with black. Use thresholding to select the resulting image containing white cell bodies and black nuclei. Add this selection to the ROI manager.
    9. Use the cell body ROI to measure the mean values of ,, , and intensity <I> from the corresponding images.
    10. Repeat the process for each image, transferring results for each parameter to Excel. For each cell type, calculate the mean (± SE) value of each parameter. Differences in parameter values can be assessed for statistical significance using a Wilcoxon signed-rank test.
    11. In each cell line, the ratio of NADPH to NADH can be related to the measured parameters using,
    12. The measured intensity and the remaining decay parameters can be used to calculate the relative concentrations of enzyme-bound NADH and NADPH using,

      Where,
      k is an arbitrary constant shared between all experiments, assuming experimental settings (e.g., laser power) were kept constant.

Notes

  1. Plating of cells for microscopy
    1. Coverslips can be sterilised prior to being placed into the 6 well plate. This can be achieved by autoclaving, passing through a flame, or being dipped in ethanol. Sufficient time should be left for the coverslips to cool or for all traces of ethanol to evaporate before the cell suspension is added to the well.
    2. Different cell lines exhibit different adherence for glass. Cells that attach poorly may require treatment of the coverslips with gelatin, fibronectin or poly-L-lysine. Coverslips can also be purchased pre-treated.
  2. NAD redox state assay
    1. The NAD(P)H fluorescence intensity is low relative to extrinsic fluorophores conventionally imaged using confocal microscopy. As such, care should be taken to fully optimise the signal prior to experimental recordings. The on-sample laser power chosen should not be so high as to cause bleaching, and associated photodamage (Tiede and Nichols, 2006), of the detected signal over the ~30 min time period of the experiment. The highest numerical aperture objective available should be chosen, perhaps necessitating the use of an oil immersion lens in an inverted configuration. Laser collimators should be adjusted to maximise contrast and confocal pinholes should be a large as possible, as precise z-resolution is not required in this assay. Finally, the detector gain and offset settings should be chosen in preliminary experiments such that FCCP does not cause the detected signal to reach zero and that rotenone does not cause detector saturation.
    2. Acquisition settings (e.g., laser power and detector gain) must be kept constant in order to obtain comparable results between technical and biological replicates. Since measured intensities are a linear function of the applied laser power with single-photon excitation, linear correction (normalisation) can be applied if the laser power is modified. Gain on confocal microscopes has a non-linear effect on intensities which makes normalisation impractical if it is adjusted. Ideally, threshold settings during analysis should also be kept constant in a given set of experiments, in particular if absolute levels of NAD(P)H are compared between different conditions or cell lines.
    3. For assistance in choice of excitation wavelength and filtering, the absorption and emission spectra of NAD(P)H are included in Figure 6, adapted from Patterson et al. (2000).


      Figure 6. Absorption and emission spectra of NAD(P)H (adapted from Patterson et al., 2000)

    4. The NAD(P)H fluorescence signal should be allowed to reach a steady level following the addition of FCCP or rotenone before the experimental conditions are subsequently altered (addition of the next drug, or termination of the experiment). This may require longer than the 8 min suggested here due to the time taken for the drug to mix around the dish. This process may be aided by pipetting larger volumes of a more dilute solution to reach the final concentration.
    5. The primary effects of FCCP and rotenone are to oxidise and reduce the NAD pool, respectively. As such, this can be viewed as an assay of the mitochondrial NAD redox state. However, these treatments may have secondary effects on the NADP pool through the action of the mitochondrial nicotinamide nucleotide transhydrogenase (NNT). This will oxidise NADH to reduce NADPH under control and rotenone-treated conditions but may reverse to consume NADPH and replenish the NADH supply with FCCP (Nickel et al., 2015). The qualitative influence on the final result will be minimal as the NAD pool is much larger than the NADP pool, but this caveat should be noted when attempting to draw quantitative conclusions (Blacker and Duchen, 2016).
    6. Once experimental protocols have been correctly established, this assay is generally highly reproducible, with typical standard deviations of the scaled fluorescence levels across identical experiments of around 2%. This allows around 3-5 repeats to be sufficient to have statistical confidence in the final results.
  3. NAD(P)H FLIM assay
    1. As the detectors required for FLIM have single photon sensitivity, their protection from bright light sources is of utmost importance. Shutter assemblies should be purchased with the detectors, blackout curtains over the microscope itself (Figure 1) should be used to protect from room lights and use of mercury lamps in the experiments should be minimised. Seals between the detector and the microscope should be routinely inspected to ensure no light can leak in.
    2. For the most accurate extraction of fluorescence decay parameters from FLIM data, SPCImage should be supplied with a measurement of the instrument response function of the system. This can be acquired by measuring the fluorescence lifetime profile of a scattering object. For example, we routinely use the second harmonic generation signal of potassium dihydrogen phosphate (KDP) crystals, grown by leaving a molar solution on a coverslip to evaporate overnight.
    3. This protocol has been written assuming two-photon excitation will be used for FLIM. Pulsed UV lasers exist, allowing NAD(P)H fluorescence lifetime measurements with single photon excitation. However, the pulsed Ti:sapphire laser is ubiquitous in biomedical microscopy facilities in order to perform two-photon intensity imaging on thick samples. As such, FLIM add-ons are typically installed onto these existing systems.
    4. The models used to interpret NAD(P)H FLIM data in terms of the separate concentrations of NADH and NADPH (equations 2-4) are subject to a number of assumptions (Blacker et al., 2014). The balance of the two cofactors in the enzyme-bound population is assumed to reflect that in the free population, and these species are assumed to possess finite, distinct lifetimes. These models have shown success in unravelling the separate roles of NADH and NADPH in a number of biological contexts (Blacker et al., 2014; Nickel et al., 2015; Tosatto et al., 2016). However, their quantitative accuracy will be increased by ongoing refinements based on enhanced understanding of the contrasting photophysics of the enzyme-bound cofactors, and their relationship to the underlying metabolism.
    5. Pixel-to-pixel variability exists in the fluorescence lifetimes measured in an NAD(P)H FLIM image due to noise inherent to the TCSPC technique. Extracting the mean lifetime value from a region of interest has been shown to negate the effect of this Poisson noise, reporting the true underlying lifetime value (Blacker et al., 2014). Cell to cell variability in the fluorescence decay parameters also exists, with standard deviations of around 5%. As such, we typically acquire 3-5 images per coverslip over 3-6 coverslips in order to assess statistically significant differences in the fluorescence decay parameters between conditions.

Recipes

  1. Routine cell culture medium (500 ml)
    440 ml Advanced DMEM (500 ml bottle with 60 ml removed)
    50 ml fetal bovine serum (10% final concentration)
    5 ml GlutaMAXTM (2 mM final concentration)
    5 ml penicillin-streptomycin (100 U ml-1 penicillin and 100 mg ml-1 streptomycin final)
  2. Live-cell imaging medium (50 ml)
    415 mg DMEM powder
    0.5 ml GlutaMAXTM (2 mM final concentration)
    225 mg D-(+) glucose (25 mM final concentration)
    5.5 mg sodium pyruvate (1 mM final concentration)
    119 mg HEPES (10 mM final concentration)
    Top up to 50 ml with ultrapure water
    Adjust pH to 7.4, increasing using NaOH or decreasing using HCl
    Sterilise using syringe filter
  3. ETC perturbations
    1. FCCP: 2.5 mg in 10 ml ethanol for 1 mM stock solution (stored in freezer). Dilute 25 μl in 5 ml of imaging medium for 5 μM working solution on day of experiment
    2. Rotenone: 3.9 mg in 10 ml ethanol for 1 mM stock solution (stored in freezer). Dilute 150 μl in 5 ml of imaging medium for 30 μM working solution on day of experiment

Acknowledgments

The use of NAD(P)H autofluorescence for assessing the redox state of live tissues was originated by Britton Chance in the 1950’s (Chance et al., 1962; Chance et al., 1952; Chance and Williams, 1955). These protocols extend this pioneering work onto modern apparatus. We acknowledge support from BBSRC grant BB/L020874/1.

References

  1. Blacker, T. S. and Duchen, M. R. (2016). Investigating mitochondrial redox state using NADH and NADPH autofluorescence. Free Radic Biol Med 100: 53-65
  2. Blacker, T. S., Mann, Z. F., Gale, J. E., Ziegler, M., Bain, A. J., Szabadkai, G. and Duchen, M. R. (2014). Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat Commun 5: 3936.
  3. Chance, B. (1952). Spectra and reaction kinetics of respiratory pigments of homogenized and intact cells. Nature 169(4293): 215-221.
  4. Chance, B., Cohen, P., Jobsis, F. and Schoener, B. (1962). Intracellular oxidation-reduction states in vivo. Science 137(3529): 499-508.
  5. Chance, B. and Williams, G. R. (1955). Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem 217(1): 409-427.
  6. De Ruyck, J., Fameree, M., Wouters, J., Perpete, E. A., Preat, J. and Jacquemin, D. (2007). Towards the understanding of the absorption spectra of NAD(P)H/NAD(P)+ as a common indicator of dehydrogenase enzymatic activity. Chem Phys Lett 450, 119-122.
  7. Duchen, M. R., Surin, A. and Jacobson, J. (2003). Imaging mitochondrial function in intact cells. Methods Enzymol 361: 353-389.
  8. Murphy, M. P. (2009). How mitochondria produce reactive oxygen species. Biochem J 417, 1-13.
  9. Nickel, A. G., von Hardenberg, A., Hohl, M., Loffler, J. R., Kohlhaas, M., Becker, J., Reil, J. C., Kazakov, A., Bonnekoh, J., Stadelmaier, M., Puhl, S. L., Wagner, M., Bogeski, I., Cortassa, S., Kappl, R., Pasieka, B., Lafontaine, M., Lancaster, C. R., Blacker, T. S., Hall, A. R., Duchen, M. R., Kastner, L., Lipp, P., Zeller, T., Muller, C., Knopp, A., Laufs, U., Bohm, M., Hoth, M. and Maack, C. (2015). Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure. Cell Metab 22(3): 472-484.
  10. Osellame, L. D., Blacker, T. S. and Duchen, M. R. (2012). Cellular and molecular mechanisms of mitochondrial function. Best Pract Res Clin Endocrinol Metab 26(6): 711-723.
  11. Patterson, G. H., Knobel, S. M., Arkhammar, P., Thastrup, O. and Piston, D. W. (2000). Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells. Proc Natl Acad Sci U S A 97(10): 5203-5207.
  12. Pawley, J. (2012). Handbook of Biological Confocal Microscopy. Springer.
  13. Pollak, N., Dolle, C. and Ziegler, M. (2007). The power to reduce: pyridine nucleotides--small molecules with a multitude of functions. Biochem J 402(2): 205-218.
  14. Tiede, L. M. and Nichols, M. G. (2006). Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy. Photochem Photobiol 82(3): 656-664.
  15. Tosatto, A., Sommaggio, R., Kummerow, C., Bentham, R. B., Blacker, T. S., Berecz, T., Duchen, M. R., Rosato, A., Bogeski, I., Szabadkai, G., Rizzuto, R. and Mammucari, C. (2016). The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1α. EMBO Mol Med 8(5): 569-585.
  16. Ying, W. (2008). NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal 10(2): 179-206.

简介

NADH和NADPH分别是分解代谢和合成代谢过程的氧化还原辅因子。此外,NADPH在细胞抗氧化防御中起着重要作用。在活细胞和组织中,其光谱相同的自发荧光(称为NAD(P)H)的强度可用于探测线粒体氧化还原状态,而其不同的酶结合特征可用于将其相对贡献与总共分离使用荧光寿命成像显微镜(FLIM)的NAD(P)H强度。这些方案允许在细胞类型和改变的生理和病理状态之间检测代谢的差异。

背景 氧化还原辅因子烟酰胺腺嘌呤二核苷酸(NADH)及其磷酸化对应物NADPH的还原形式本质上是荧光的,两者都吸收波长为340(±30)nm并在460(±50)nm处发射的光(Patterson等人。,2000)。这些光谱特征在氧化成NAD(上标+)或NADP(superson),(2007))时损失。单独的NAD和NADP池的氧化还原平衡决定了对比的代谢过程(Ying,2008),如图1所示。NAD作为电子受体,用于通过三羧酸氧化线粒体中的糖,脂质和氨基酸底物(TCA)循环,并作为内线粒体膜(IMM)上的电子传递链(ETC)的电子供体,促使将质子泵送到膜间隙中,作为合成三磷酸腺苷(ATP)的电源,通过F 1 F 0 O 3 ATP合成酶(Osellame等人,2012)。因此,线粒体中NADH与NAD + 的平衡反映了TCA循环与ETC活性的平衡。 ETC功能障碍导致NADH / NAD + 比例的增加和潜在有害的活性氧(ROS)的产生(Murphy,2009)。细胞的抗氧化防御要求NADP池为其维持提供减少的等同物,因此NADPH / NADP + 比必须保持较高(> 3)(Pollak等人。 ,2007)。因此,线粒体NAD池的氧化还原状态和NADPH的相对丰度是细胞类型氧化应激水平的关键因素。


图1.线粒体NAD(P)H代谢的示意图。TCA循环中的底物氧化将电子转移到NAD +,形成NADH。 A.在静止条件下,NADH携带的电子沿着ETC通过,为从线粒体基质穿过内部线粒体膜(IMM)泵送质子提供给间隔空间。所得到的质子梯度对ETC(F 1 O 3 O 3 O 3 O ATP合酶)的复合物V的ATP产生产生作用。 B.加入解偶联剂如羰基氰化物4-(三氟甲氧基)苯腙(FCCP)允许质子在IMM上回漏,导致ETC处的NADH的氧化速率增加以恢复膜梯度。 C.通过鱼藤酮对ETC的抑制使NADH氧化并导致线粒体ROS的近端来源超氧化物(O 2)的产生增加。这被NADPH维持的超氧化物歧化酶(SOD2)和谷胱甘肽(GSH / GSSG)抗氧化防御系统转化成水被中和。

&nbsp;这里,我们描述了评估线粒体NADH / NAD + 比例和NADPH / NADH平衡依赖于这些辅因子的荧光减少时的方案。它们相同的吸收和发射光谱导致组合信号被称为NAD(P)H(Blacker和Duchen,2016)。在应用ETC解偶联剂和抑制剂后,使用共焦显微镜测量NAD(P)H荧光的变化允许估计线粒体NADH / NAD +平衡(Duchen等人,,2003)。为了区分NADH和NADPH对总信号的相对贡献,必须引入荧光寿命成像显微镜(FLIM)(Blacker等人,2014)。这些方案更详细地描述了在Tosatto等人(2016)中使用的方法来研究负责线粒体钙摄取的选择性通道(线粒体钙单通道(MCU))在进展中的作用的乳腺癌。假设共焦显微镜的基本理解。作为背景,读者将被引导至Pawley等人。(2012)。

关键字:NADH, NADPH, NAD(P)H, 自体荧光, 显微镜检查, 荧光寿命, FLIM, 氧化还原状态

材料和试剂

  1. Nunc TM细胞培养处理的EasYFlasks TM(75cm 2,过滤器封闭)(Thermo Fisher Scientific,Thermo Scientific TM >,目录号:156499)
  2. Falcon管(15毫升)(VWR,目录号:734-0451)
  3. 提示
  4. Falcon管(50ml)(VWR,目录号:734-0448)
  5. Eppendorf管(1.5ml)(VWR,目录号:211-2520)
  6. (6孔)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)的Nunc TM 细胞培养处理的多片
  7. 盖板(22mm,厚度No.1)(VWR,目录号:631-0158)
  8. 注射器(20 ml,VWR,目录号:613-3921)
  9. 过滤灭菌器(0.2μm,VWR,目录号:514-0064)
  10. MDA-MB-231细胞(ATCC,目录号:HTB-26)
  11. 先进的Dulbecco改良Eagle's培养基(DMEM)(500ml)(Thermo Fisher Scientific,Gibco TM,目录号:12491023)
  12. 胎牛血清(FBS,热灭活,50ml)(Thermo Fisher Scientific,Gibco TM,目录号:10500064)
  13. (5ml)(Thermo Fisher Scientific,Gibco TM,目录号:35050061)
  14. 青霉素 - 链霉素(10,000U ml -1),5ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  15. 胰蛋白酶-EDTA(0.25%,酚红,100ml)(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  16. FCCP(10mg)(Sigma-Aldrich,目录号:C2920)
  17. 鱼藤酮(5g)(Sigma-Aldrich,目录号:R8875)
  18. 乙醇(1L)(Sigma-Aldrich,目录号:02860)
  19. Dulbecco改良的Eagle's中等粉末(无葡萄糖,L-谷氨酰胺,酚红,丙酮酸钠和碳酸氢钠)(Sigma-Aldrich,目录号:D5030)
  20. D-(+)葡萄糖(100g)(Sigma-Aldrich,目录号:G8270)
  21. 丙酮酸钠(25g)(Sigma-Aldrich,目录号:P2256)
  22. HEPES(100g)(Sigma-Aldrich,目录号:H3375)
  23. 氢氧化钠溶液(1N,1L)(Sigma-Aldrich,目录号:71463)
  24. 盐酸溶液(1N,1L)(Sigma-Aldrich,目录号:71763)
  25. 常规细胞培养基(500毫升)(见配方)
  26. 活细胞成像培养基(50ml)(参见食谱)
  27. ETC扰动(见配方)

设备

  1. 培养箱(37℃,5%CO 2)(Thermo Fisher Scientific,Thermo Scientific,superson TM,型号:HERAcell TM 150) >
  2. 离心机配有15 ml Falcon管容量(Mega Star 1.6)(VWR,目录号:521-1749)
  3. 血细胞计数器(Neubauer)(VWR,目录号:630-1509)
  4. 激光扫描显微镜(Zeiss,型号:LSM510)
    1. 40倍放大目标
    2. 遮阳窗帘,房间和覆盖FLIM显微镜(见图2)



      图2.高信噪比FLIM的设备。 A.具有双光子激发的相干变色龙的蔡司LSM510和Becker& Hickl HPM-100-40检测器和SPC830计数电子元件。 B.显微镜室和显微镜本身周围的遮光窗帘,用于确保FLIM影像中的背景噪音保持在最低水平。

    3. NAD(P)H荧光发射滤光片(435-485 nm)
    4. 对于单光子激发:
      1. 351 nm激光源(Enterprise UV,Coherent)
      2. 长通二色(375 nm截止)
      3. 石英显微镜光学
    5. 对于双光子激发:
      1. Ti:蓝宝石激光器以720nm模式锁定(Chameleon Ultra,Coherent)
      2. 短路二色(650nm截止)
      3. 非下行检测
    6. 对于FLIM:
      1. 具有单光子灵敏度的检测器(Becker& Hickl,型号:HPM-100-40)
      2. 时间相关单光子计数(TCSPC)电子学(Becker& Hickl,型号:SPC-830)
      3. 脉冲激发源(双光子激发由Ti:蓝宝石激光器在720nm;变色龙Ultra,Coherent)
    7. 加热显微镜台(定制保持成像环,见图3)



      图3.用于在显微镜上安装盖玻片的设备。 A.定制环和用于固定22毫米圆形盖玻片的安装块。 B.盖片直接放在金属环的底部上。 C.具有橡胶密封件的同心环放置在盖玻片的顶部。 D.将第三个环拧到前两个上,以便在盖玻片上形成防水密封,允许(E)成像缓冲液移液到细胞上。 F.成像环和细胞然后适合加热显微镜阶段。

  5. 直头钳(VWR,目录号:232-0094)
  6. 成像环(定制,见图3)
  7. 倒置相位对比度台式显微镜(VWR,目录号:630-2145; ZEISS,型号:Primovert)
  8. 实验室平衡(0.1mg分辨率)(Mettler-Toledo,目录号:30029067)
  9. pH计(Mettler-Toledo International,目录号:30266626)
  10. 计数器(VWR,目录号:720-1984)

软件

  1. 显微镜控制软件(Zeiss ZEN)
  2. FLIM采集软件(Becker& Hickl SPCM)
  3. FLIM配件软件(Becker& Hickl SPCImage)
  4. 图像分析软件(NIH ImageJ)

程序

  1. 常规培养MDA-MB-231细胞
    1. 培养物控制(杂交质粒)和单克隆抗体敲低MDA-MB-231细胞,在补充有10%FBS,2mM GlutaMAX ,100U/ml青霉素的Advanced DMEM中, 100μg/ml链霉素。
    2. 在37℃,5%CO 2培养箱中,在含有10-12ml生长培养基的75cm 2的组织培养瓶中保持细胞作为单层。
    3. 通过在70-80%汇合时以1:10的比例分裂来保证每3-4天的通过。
    4. 通过用2ml胰蛋白酶的初步洗涤分离细胞,然后与2ml新鲜胰蛋白酶孵育5分钟。

  2. 电镀细胞进行显微镜检查
    1. 从75 cm 2烧瓶中弃去生长培养基,并用3 ml胰蛋白酶孵育5 min,分离细胞。
    2. 通过加入5ml生长培养基收集细胞并抑制酶。
    3. 将烧瓶的内容物转移到15ml Falcon管中,并在室温(24℃)下以400×g离心5分钟。
    4. 将细胞重悬于5ml新鲜培养基中,并将少量(〜100μl)转移至Eppendorf管进行细胞计数。
    5. 在台式显微镜上使用血细胞计数器和计数器计数再悬浮细胞浓度,确保将Eppendorf管的内容物与移液管充分混合以解决沉淀。
    6. 使用细胞计数结果,将来自细胞悬浮液的1,800,000个细胞移液到新的50ml Falcon管中(每孔30孔细胞6孔板)。加满管,总共含有12ml(每孔2ml总培养基)。
    7. 使用镊子,将一个盖玻片转移到6孔板的每个孔中。
    8. 将2ml的180万个细胞混合物移入每个孔中,再次确保在转移之前使用移液管混合管的内容物。
    9. 将6孔板置于培养箱中,放置过夜,以便在第二天成像之前将细胞附着到盖玻片上。
    10. 在分离的6孔板中对每种细胞类型进行重复过程。

  3. NAD氧化还原状态测定
    1. 使用镊子将盖玻片从6孔板转移到金属成像环中(见图3)。拧紧直到感觉到少量阻力以确保防水密封。
    2. 轻轻洗涤细胞(确保细胞未分离)将400μl成像缓冲液移至盖玻片上,以除去剩余的酚红。
    3. 处理洗涤介质,轻轻移液400μl成像缓冲液至细胞上。
    4. 将盖玻片转移到共聚焦显微镜的加热阶段,并在明场照明下使细胞聚焦。
    5. 使用375nm长的二向色和435-485nm发射滤光片,逐渐增加351nm激发激光的强度,直到清楚地观察到NAD(P)H荧光。
    6. 搜索盖玻片并放大图像,使得在视野中可以观察到10-20个细胞。
    7. 开始时间序列采集,每2分钟成像一次。
    8. 4张图像(从时间序列开始6分钟)后,轻轻地将100μl5μMFCCP溶液直接加入到细胞上,给出最终的FCCP浓度为1μM。由于增加的ETC活性,NAD(P)H荧光信号应开始降低。
    9. 在FCCP处理(加入8分钟)后获得4张图像,轻轻地将100μl30μM鱼藤酮溶液直接加入到细胞上,得到最终的5%的鱼藤酮浓度。由于抑制ETC,NAD(P)H荧光信号应开始增加。
    10. 在鱼藤酮加入10分钟后终止实验,总试验时间为24分钟。
    11. 处理细胞并使用100%乙醇清洁可能与FCCP或鱼藤酮(例如成像环,显微镜物镜)接触的任何实验设备。
    12. 重复所有盖玻片的程序,并保存数据供后续分析。

  4. NAD(P)H FLIM测定
    1. 在金属成像环中用400μl成像缓冲液(与氧化还原状态测定相同)准备盖玻片,并在显微镜上放置,调整焦点以在明场照射下观察细胞。
    2. 使用650nm的短路二色性和435-485nm的发射滤光片,逐渐增加720nm激发激光的强度,直到用显微镜的内部检测器观察到NAD(P)H荧光。
    3. 搜索盖玻片并放大图像,使得在视野中可以观察到10-20个细胞。
    4. 调整显微镜设置,将荧光信号发送到FLIM探测器所在的非下行端口。
    5. 开始FLIM数据采集,扫描2分钟。
    6. 移动到盖玻片上的新位置并重复过程。每个盖玻片获取3-5张图像。
    7. 处理细胞并使用100%乙醇清洁可能与FCCP或鱼藤酮(例如成像环,显微镜物镜)接触的任何实验设备。
    8. 重复所有盖玻片和细胞类型的步骤。

数据分析

  1. NAD氧化还原态测定(参见图4)


    图4.线粒体NAD氧化还原状态测定的示意图。 使用1μMFCCP和1μM鱼藤酮处理后的荧光强度作为动态范围,通过该动态范围可以评估降低的NADH的静息荧光强度,从而降低NADH的静息百分比。阈值用于选择高于定义的亮度值(,即,包含单元格的像素)的像素,减少背景噪声的影响。

    1. 将时间序列图像导入ImageJ。数据集将被表示为"堆栈",x轴和y轴表示图像的空间坐标,z轴表示自实验开始以来的时间。可以通过移动水平滚动条导航时间序列。
    2. 使用阈值工具以红色高亮显示单元格区域。应选择最小值和最大值,使得在任何时间点没有选择背景信号,并且不应该忽略明亮的细胞内区域(例如,鱼藤酮)。
    3. 通过在"设置测量"对话框中单击"限制到阈值",确保测量被限制到阈值区域,否定背景荧光的影响。
    4. 通过从Stacks菜单中选择Plot Z轴曲线绘制NAD(P)H荧光信号的时间演变。将数据导出到Microsoft Excel。
    5. 对每个时间序列采集重复此过程。
    6. 在Excel中,通过应用以下公式,将每个时间点F(t)的浓度按其时间序列的最小值(含FCCP)和最大值(与鱼藤酮值)进行比较: br />
    7. 该过程的正确应用将使每个时间序列的最小值为0%,最大值为100%。
    8. 对于每个细胞系,采取在静息条件下获得的所有缩放数据点的平均值,以计算最终的NADH(%)值。将不确定性表示为这些要点的标准误差。每个细胞系的NADH(%)值之间的任何差异的统计学意义可以使用Wilcoxon有符号秩检验进行评估。
  2. NAD(P)H FLIM测定(参见图5)


    图5. FLIM分析软件。在SPCImage(A)中执行双指数荧光衰减到FLIM图像的拟合。参数矩阵输出到ImageJ(B)进行后续分析。通过组合通过阈值产生的整个细胞的掩模,并排除手动绘制的核区域,产生含有细胞溶质和线粒体的感兴趣区域。

    1. 在SPCImage中,导入FLIM图像并调整设置以在每个像素处适合2个衰减分量。
    2. 为了确保足够的准确性,应该增加合并,直到在感兴趣的最暗的胞质像素中的衰减峰值中包含超过100个计数(Blacker等人,2014)。对于激光功率保持足够低以避免电池损坏的2分钟采集,这通常需要2到4的合并因子,对应于来自24和80周围像素之间的数据的总和。
    3. 通过选择计算衰减矩阵开始每个像素的拟合过程。这个过程可能需要5分钟,这取决于所使用的计算机的功率。
    4. 导出每个衰减参数的矩阵 - <2> (%),  - 光子计数每个像素用于ImageJ中的后续分析。
    5. 导入α 2 (%),< img width ="10" height ="11" alt =""src ="/attached/image/20170119/20170119000426_9254.jpg"/>, 并且使用导入文本图像命令将光子计数图像转换为ImageJ。
    6. 在光子计数图像上使用阈值和创建选择命令,将包含单元格的感兴趣区域(ROI)添加到ROI管理器。
    7. 使用多边形选择工具创建单元格核的投资回报率并保存到投资回报率管理器。由于代谢与细胞其余部分的差异,核通常具有改变的NAD(P)H荧光衰减特性,因此为了简单起见,这里忽略了这些分析。
    8. 从单元格ROI创建一个蒙版。在掩码窗口中选择核ROI,并填充黑色。使用阈值来选择包含白色单元体和黑色核的结果图像。将此选择添加到ROI管理器。
    9. 使用细胞体ROI测量平均值,和强度 I>
    10. 对每个图像重复该过程,将每个参数的结果传输到Excel。对于每个单元格类型,计算每个参数的平均值(±SE)值。使用Wilcoxon有符号秩检验可以评估参数值的差异,以获得统计学显着性。
    11. 在每个细胞系中,NADPH与NADH的比例可以与使用
      的测量参数相关
    12. 测量的强度和剩余的衰变参数可用于计算酶结合的NADH和NADPH的相对浓度,使用,

      哪里,
      假设实验设置(例如,激光功率)保持恒定,所有实验之间共享任意常数。

笔记

  1. 电镀细胞进行显微镜检查
    1. 盖板可以在放入6孔板之前进行灭菌。这可以通过高压灭菌,通过火焰或浸入乙醇来实现。留下足够的时间让盖玻片冷却,或者在将细胞悬浮液加入到孔中之前,所有痕量的乙醇蒸发。
    2. 不同的细胞系对玻璃表现出不同的粘附性。附着不良的细胞可能需要用明胶,纤连蛋白或聚-L-赖氨酸处理盖玻片。盖片也可以预先处理。
  2. NAD氧化还原状态测定
    1. 相对于使用共焦显微镜常规成像的外在荧光团,NAD(P)H荧光强度较低。因此,应注意在实验记录之前对信号进行全面优化。在实验的〜30分钟时间内,所选择的采样激光功率不应该高到引起漂白和相关的光损伤(Tiede和Nichols,2006)的检测信号。应选择可用的最高数值孔径物镜,这可能需要使用倒置配置的油浸透镜。应该调整激光准直器以最大化对比度,并且共聚焦针孔应尽可能大,因为该测定中不需要精确的z分辨率。最后,在初步实验中应选择检测器增益和偏移设置,使得FCCP不会使检测到的信号达到零,并且鱼藤酮不会导致检测器饱和。
    2. 必须保持采集设置(例如,,激光功率和检测器增益),以便在技术和生物重复之间获得可比较的结果。由于测量的强度是使用单光子激发施加的激光功率的线性函数,所以如果激光功率被改变,则可以应用线性校正(归一化)。共焦显微镜上的增益对强度有非线性影响,如果调整,则使归一化不切实际。理想情况下,分析中的阈值设置也应在给定的一组实验中保持不变,特别是如果在不同条件或细胞系之间比较绝对水平的NAD(P)H。
    3. 为了帮助选择激发波长和过滤,NAD(P)H的吸收和发射光谱包括在图6中,改编自Patterson等人(2000)。


      图6. NAD(P)H 的吸收和发射光谱(改编自Patterson等人,2000)

    4. 在添加FCCP或鱼藤酮后,NAD(P)H荧光信号应允许达到稳定水平,然后在实验条件随后改变之前(添加下一种药物或终止实验)。这可能需要比这里建议的8分钟更长的时间,因为药物在盘子周围混合所花费的时间。通过吸取更大体积的更稀释溶液以达到最终浓度,可以帮助该过程。
    5. FCCP和鱼藤酮的主要作用是分别氧化和还原NAD池。因此,这可以被视为线粒体NAD氧化还原状态的测定。然而,这些治疗可能通过线粒体烟酰胺核苷酸转氢酶(NNT)的作用对NADP池具有继发作用。这将氧化NADH以在对照和鱼藤酮处理的条件下还原NADPH,但是可以逆转消耗NADPH并用FCCP补充NADH供应(Nickel等人,2015)。由于NAD池比NADP池大得多,因此对最终结果的定性影响将是最小的,但是当试图得出定量结论时应注意这一点(Blacker和Duchen,2016)。
    6. 一旦实验方案已被正确建立,该测定通常是高度可重复的,通过相同实验的标度荧光水平的典型标准偏差约为2%。这允许约3-5次重复足以对最终结果具有统计信心。
  3. NAD(P)H FLIM测定
    1. 由于FLIM所需的检测器具有单光子灵敏度,因此它们对亮光源的保护至关重要。快门组件应与检测器一起购买,显微镜本身上的遮光窗帘(图1)应用于保护室内灯光,并且应在实验中使用汞灯。检查器和显微镜之间的密封应经常检查,以确保没有光线可能泄漏。
    2. 为了从FLIM数据中最精确地提取荧光衰减参数,应向SPCImage提供系统仪器响应功能的测量值。这可以通过测量散射物体的荧光寿命分布来获得。例如,我们通常使用磷酸二氢钾(KDP)晶体的二次谐波生成信号,通过在盖玻片上留下摩尔溶液蒸发过夜而生长。
    3. 该协议已被写入,假设双光子激发将用于FLIM。脉冲紫外激光器存在,允许使用单光子激发的NAD(P)H荧光寿命测量。然而,脉冲Ti:蓝宝石激光在生物医学显微镜设备中是普遍存在的,以便在厚样品上进行双光子强度成像。因此,FLIM附加组件通常安装在这些现有系统上。
    4. 根据NADH和NADPH的不同浓度(方程2-4),用于解释NAD(P)H FLIM数据的模型受许多假设的约束(Blacker等人,2014) 。假设酶结合人群中两个辅因子的平衡反映了在自由人群中,这些物种被假设为具有有限的,不同的生命周期。这些模型已经显示出在许多生物环境中解开NADH和NADPH的独立作用的成功(Blacker等人,2014; Nickel等人,2015; Tosatto 等,,2016)。然而,通过对增强对酶结合辅因子的对比光物理学的理解以及它们与潜在代谢的关系的进一步改进,它们的定量精确度将会增加。
    5. 由于TCSPC技术固有的噪声,在NAD(P)H FLIM图像中测量的荧光寿命中存在像素到像素的变异性。从感兴趣的区域提取平均生命周期价值已显示否定了这种泊松噪声的影响,报告了真正的潜在寿命价值(Blacker等人,2014)。荧光衰减参数中的细胞与细胞变异性也存在,标准偏差约为5%。因此,我们通常在3-6个盖玻片上获得每个盖玻片3-5个图像,以评估条件之间的荧光衰减参数的统计学显着差异。

食谱

  1. 常规细胞培养液(500 ml)
    440毫升高级DMEM(500毫升瓶,60毫升去除)
    50ml胎牛血清(10%终浓度)
    5ml GlutaMAX (2mM终浓度)
    5毫升青霉素 - 链霉素(100毫升青霉素和100mg毫升-1链霉素最终)
  2. 活细胞成像培养基(50ml)
    415mg DMEM粉末 0.5ml GlutaMAX (2mM终浓度)
    225 mg D - (+)葡萄糖(25 mM终浓度)
    5.5毫克丙酮酸钠(1毫摩尔终浓度)
    119 mg HEPES(10 mM终浓度)
    用超纯水
    加满50毫升 调节pH至7.4,使用NaOH增加或使用HCl减少
    使用注射器过滤器进行杀菌
  3. ETC扰动
    1. FCCP:将2.5mg在10ml乙醇中的1mM储备溶液(储存在冷冻器中)。在实验一天,在5ml成像培养基中稀释25μl5μM工作溶液
    2. 鱼藤酮:在10ml乙醇中用于1mM储备液(储存在冷冻箱中)3.9mg。在实验一天,在5ml成像介质中稀释150μl30μM工作溶液

致谢

使用NAD(P)H自发荧光来评估活组织的氧化还原状态是由Britton Chance于1950年代发起的(Chance等人,1962; Chance等人。 >,1952; Chance和Williams,1955)。这些协议将这一开创性工作扩展到现代化设备上。我们承认BBSRC授权BB/L020874/1的支持。

参考文献

  1. Blacker,TS和Duchen,MR(2016)。调查线粒体氧化还原状态使用NADH和NADPH自发荧光。免费Radic Biol Med 100:53-65
  2. Blacker,TS,Mann,ZF,Gale,JE,Ziegler,M.,Bain,AJ,Szabadkai,G.and Duchen,MR(2014)。  使用FLIM在活细胞和组织中分离NADH和NADPH荧光。 Nat Commun 5:3936 。
  3. Chance,B.(1952)。  光谱和反应动力学的均质和完整细胞的呼吸颜料。自然169(4293):215-221。
  4. Chance,B.,Cohen,P.,Jobsis,F. and Schoener,B.(1962)。  体内细胞内氧化还原状态 科学 137(3529):499-508。 >
  5. Chance,B. and Williams,GR(1955)。  呼吸酶氧化磷酸化。三,稳定状态。 J Biol Chem 217(1):409-427。
  6. De Ruyck,J.,Fameree,M.,Wouters,J.,Perpete,EA,Preat,J.and Jacquemin,D。(2007)。  了解NAD(P)H/NAD(P)的吸收光谱作为常见的脱氢酶酶活性的指标。化学物理学实验室,450,119-122。
  7. Duchen,MR,Surin,A.and Jacobson,J。(2003)。  在完整细胞中成像线粒体功能。 方法Enzymol 361:353-389。
  8. Murphy,MP(2009)。  线粒体如何产生活性氧物质。生物化学J 417,1-13。
  9. Nickel,AG,von Hardenberg,A.,Hohl,M.,Loffler,JR,Kohlhaas,M.,Becker,J.,Reil,JC,Kazakov,A.,Bonnekoh,J.,Stadelmaier,M.,Puhl, SL,Wagner,M.,Bogeski,I.,Cortassa,S.,Kappl,R.,Pasieka,B.,Lafontaine,M.,Lancaster,CR,Blacker,TS,Hall,AR,Duchen,MR,Kastner, L.,Lipp,P.,Zeller,T.,Muller,C.,Knopp,A.,Laufs,U.,Bohm,M.,Hoth,M。和Maack,C.(2015)。线粒体转氢酶的反转引起心力衰竭中的氧化应激。 em> Cell Metab 22(3):472-484。
  10. Osellame,LD,Blacker,TS和Duchen,MR(2012)。  生物共聚焦显微镜手册。 Springer 。
  11. Pollak,N.,Dolle,C.和Ziegler,M。(2007)。 减少能力:吡啶核苷酸 - 具有多数的小分子的功能。生物化学J 402(2):205-218。
  12. Tiede,LM和Nichols,MG(2006)。  Photobleaching降低的烟酰胺腺嘌呤二核苷酸和多光子显微镜中大鼠嗜碱性白血病细胞中高度荧光病变的发展。 Photochem Photobiol 82(3):656-664。
  13. Tosatto,A.,Sommaggio,R.,Kummerow,C.,Bentham,RB,Blacker,TS,Berecz,T.,Duchen,MR,Rosato,A.,Bogeski,I.,Szabadkai,G.,Rizzuto,R 。和Mammucari,C。(2016)。线粒体钙单通道调节通过HIF-1α的乳腺癌进展。 EMBO Mol Med 8(5):569-585。
  14. Ying,W.(2008)。 NAD + /NADH和NADP + /NADPH在细胞功能和细胞死亡中的作用:调节和生物学后果抗氧化氧化还原信号10(2): 179-206。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Blacker, T. S., Berecz, T., Duchen, M. R. and Szabadkai, G. (2017). Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime. Bio-protocol 7(2): e2105. DOI: 10.21769/BioProtoc.2105.
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