2.1.

Cell Lines and Culture

MCF10A cells are an immortalized, nontransformed epithelial cell line derived from human fibrocystic mammary tissue. These cells are defined as precancerous breast cells; they have a near-diploid karyotype and are dependent on exogenous (serum) growth factors for proliferation.^45,46 MCF10A cells were obtained from the American Type Culture Collection and cultured as described.⁴⁵ Prior to inhibitor additions, MCF10A cells were plated onto cover glass bottomed Petri dishes 22 mm in diameter. All incubation processes for all cells were performed in an incubator type Forma Series II Water Jacketed ${\mathrm{CO}}_2$ Incubator from Thermo Scientific (Waltham, Massachusetts), which was set to 37°C and with the injection of 5% ${\mathrm{CO}}_2$ gas [food grade from The British Oxygen Company (BOC)]. All used chemicals, including buffers and solvents, were purchased from Sigma-Aldrich Pty Ltd. (Sydney, New South Wales, Australia) unless otherwise noted.

2.2.

Effect of CCCP on NAD(P)H Lifetime

MCF10A cells were cultured on sterile coverslips of Petri dishes for three days before the treatment with CCCP (C2759 from Sigma-Aldrich). As a culture medium, we used mammary epithelial basal medium (MEBM) (Lonza, Basel, Switzerland) supplemented with 3% horse serum in the first experiment and serum free in the other. Cell suspension ($100\mu \mathrm{l}$) was added to the center of each dish and incubated at 37°C. After 24 h, 1 ml of fresh medium was added for each dish and cells were incubated for another two days. After that, CCCP (50 mM stock solution in dimethyl sulfoxide (DMSO)) was prepared: $10\mu \mathrm{l}$ was added to 10 ml of the medium to attain a final concentration of $50\mu \mathrm{M}$. Then each dish in the treatment group received 1 ml of CCCP, with controls receiving only the medium. FLIM data were collected from each dish 5, 10, 15, 20, 25, 30, and 60 min after treatment. Data were collected at 60-s acquisition times at 740-nm excitation.

2.3.

Effect of Other Inhibitors on NAD(P)H Lifetime

MCF10A cells were cultured on glass coverslips for two days before treatment as described for CCCP treatments above. Coverslips were placed in plastic tissue culture Petri dishes (seven coverslips in each Petri dish), and $100\mu \mathrm{l}$ of cell suspension was added to the center of each coverslip followed by incubation at 37°C for up to 48 h. After 1, 2, 24, and 48 h, three coverslips from the treated group (with one of the inhibitors below) and one from the control group were washed three times with Hank’s balanced salt solution (HBSS, Invitrogen, Carlsbad, California) and mounted on concave slides containing $\sim 50\mu \mathrm{l}$ HBSS. With such protocol, all treated and control samples were at the same confluences and were grown in the same serum-rich or under starvation conditions to detect differences only due to inhibitor treatment. FLIM data were collected for the slides of control and treated groups. The acquisition time for the cells was 90 s (more details in Sec. 2.6) to prevent cell damage or photobleaching. The inhibitors listed below were used.

2.4.

Mitochondrial Membrane Potential Experiment

The cells were treated with $50\mu \mathrm{M}$ CCCP (C2759 from Sigma-Aldrich). After CCCP treatment of all dishes, the JC-1 stain (Life Technologies, Carlsbad, California) was used for labeling cells treated with CCCP and controls. A $200\text{-}\mu \mathrm{M}$ of JC-1 stock solution was prepared by dissolving the contents of one vial in $230\mu \mathrm{l}$ of DMSO provided. Cells were incubated with JC-1 at a final concentration of $2\mu \mathrm{M}$, followed by incubation for 20 min and washing in phosphate-buffered saline (PBS). Labeled cells were imaged using the Leica SP2 confocal microscope. For each group of control and treated cells, spectral images were collected at 488-nm excitation wavelength and detected in 10-nm windows in the 520- to 660-nm range. The collected data were unmixed to produce corrected spectra with peak emissions at 535 nm (green) and 590 nm (red), corresponding to monomeric and aggregated forms of JC-1, respectively. Unmixed images at these wavelengths were extracted and used to quantify the ratio between red and green fluorescence using the InCell Developer software (GE Healthcare, Albany, New York) to evaluate the mitochondrial membrane potential.

2.5.

Extracellular Lactate Assay

A Cayman’s Glycolysis Cell-Based Assay Kit (Abcam, United Kingdom) was used. This kit provides a colorimetric method for detecting extracellular L-lactate, the end product of nonoxidative glucose metabolism. Cells were cultured in a 96-well plate at a density of ${10}^4$ to ${10}^5\text{cells}/\text{well}$ in $120\mu \mathrm{L}$ of culture medium and incubated overnight. On the next day, cells in wells were treated with inhibitors for the same incubation times used for FLIM imaging. The kit standards and reaction solutions were prepared according to the kit protocol. From each well, $10\mu \mathrm{l}$ of cells supernatant was added to $90\mu \mathrm{l}$ of assay buffer in a new 96-well plate, followed by the addition of $100\mu \mathrm{l}$ of reaction solutions. A 96-well plate was then incubated with gentle shaking on an orbital shaker for 30 min at room temperature. The absorbance was measured at 490 nm using a Spectra MAX M5 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, California). The concentrations of L-lactate were calculated from absorbance measurements, after collecting calibration curve using standards provided with the glycolysis kit.

2.6.

FLIM Imaging

Multiphoton FLIM images were acquired on a Leica SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with Ti:sapphire mode-locking cavity laser (Spectra-Physics-Millennium-Tsunami, Santa Clara, California, 80 MHz, 100 ps) pumped by a frequency doubled YAG laser (532 nm) and the lifetime signal was detected using a fast photon counting detector (PMH-100, Becker & Hickl GmbH, Berlin, Germany). The excitation wavelength was set to 740 nm with average laser intensity of 7.8 mW and a narrow band-pass filter $452/45\mathrm{nm}$ was used to collect only the autofluorescence signal specific to NAD(P)H. Multiphoton FLIM images were acquired with an electronic system single photon counting module (SPCM) for recording fast light signals by time-correlated single-photon counting (SPC-830, Becker & Hickl GmbH). Synchronized fluorescence lifetime data collection on a pixel-by-pixel basis was achieved using the $x$ and $y$ laser scanning signals generated by the confocal scan unit with a pixel dwell time of $4.9\mu \mathrm{s}$. FLIM data were collected through a $60\times$ oil objective ($\mathrm{NA}=1.3$) with collection times ranging from 60 to 180 s during optimization to provide enough photons and prevent the cells’ degradation. For all the measurements, 90 s was chosen as it produced sufficient intensity signal for data analysis, and a cell viability test showed that the cells were not affected. FLIM measurements were collected for healthy, viable, dividing cells at high confluences, but only from regions of cells forming a single layer and always at the same cross-section plane. This cross-section plane was established before each experimental day by test measurements of Fluoresbrite YG (10 micron) microspheres (from Polysciences, Warrington, Pennsylvania); this was also to test the accuracy and consistency of the FLIM system. The measured lifetime for these control beads was $2.19\pm 0.02\mathrm{ns}$, in agreement with previous works.¹⁵

2.7.

Analysis of the Fluorescence Lifetime Decay Curves

Time-decay FLIM data were fitted with a biexponential model using the vendor supplied software (SPCImage, v4.0.8.0, Becker & Hickl GmbH). First, the measured lifetime decay curve was binned over the pixel of interest and the eight nearest-neighbor pixels. Each set of binned pixels was fitted independently. The measured instrument response was mathematically removed from the measured data using deconvolution. This procedure yielded lifetime decay curves with peak values $>100$ counts. These lifetime decay curves for a given set of binned pixels were fitted to a double-exponential decay model.

Fig. 1

Screenshot from SPCMImage software (Becker & Hickl Gmbh, Germany) of analyzed fluorescence lifetime imaging microscopy (FLIM) image of MCF10A cells untreated. (a) Intensity image. (b) Color-coded image of long lifetime component distribution. (c) Histogram of calculated long component ${\tau }_2$ of lifetime for image (a). (d) Decay curve of multiexponential decay calculated with two component lifetime contributions, blue points—experimentally measured lifetimes, green curve—instrument response function, and red curve—calculated curve fit to experimental decay.

3.1.

Multiphoton FLIM Imaging and Effect of Inhibitors on NAD(P)H Lifetime

Figure 1 shows an example of a multiphoton FLIM image of untreated MCF10A control cells and its analysis results for a binned pixel showing a good fit of the measured lifetime data to the biexponential model. The lifetime distribution in MCF10A cells shows the shortest NAD(P)H lifetimes in the nucleus and mitochondria and longer lifetimes for NAD(P)H in the cytosol, in agreement with previous observations from rodent cells.¹⁵ Here we show that the same relationship is found for human MCF10A breast cells.

3.2.

Effect of 2-Deoxy-D-Glucose on NAD(P)H Fluorescence Lifetime

FLIM images were collected from untreated MCF10A cells (controls) and treated with ($25\mu \mathrm{M}$) 2-DG in the presence and absence of serum. The extracted data are presented in Figs. 2(a) and 2(b) for different incubation times. Note that our concentration of 2-DG is around three orders of magnitude lower than is commonly employed.^27,47 For all measured samples, lifetimes for free and bound NAD(P)H were in the following ranges: ${\tau }_1$ in 0.36 to $0.52\pm 0.005\mathrm{ns}$, ${\tau }_2$ in 2.0 to $2.7\pm 0.05\mathrm{ns}$, respectively, with free-to-bound NAD(P)H ratios in the range of 1.6 to $2.8\pm 0.05$ and average lifetimes ${\tau }_{\mathrm{m}}$ in 0.9 to $1.3\pm 0.05\mathrm{ns}$ range. After the inhibitor treatment, the average lifetimes were slightly longer than in the untreated samples. The samples without serum trended toward insignificantly higher average lifetimes and longer lifetimes for free and bound NAD(P)H than the samples with serum. In the medium supplemented with 3% serum, all treated cells also trended toward higher average lifetimes compared to controls. Statistically significant changes ($p>0.05$) were observed only for a 2-h incubation period with the inhibitor for the sample grown in the medium with serum. This increase in average lifetimes was also accompanied by a significant decrease in free-to-bound NAD(P)H ratios for 2-DG-treated cells in serum observed for 1-, 2-, and 48-h incubation times. A significant decrease in the free:bound NAD(P)H ratio was observed in cells grown without serum and incubated with the inhibitor for 2 and 48 h.

Fig. 2

Summary of lifetime measurements using FLIM for MCF10A cells grown with inhibitors measured at 1, 2, 24, and 48 h after adding the inhibitor and compared with their controls grown for the same period of time but without inhibitors in two medium types. Shaded columns correspond to MCF10A cells grown in medium without serum and white columns for cells grown with 3% serum and are shown for controls (−) and treated samples with inhibitors (+), respectively. Left column summarizes average lifetimes and right column shows free to bound NAD(P)H ratios for samples grown with following inhibitors: 2-deoxy-D-glucose (2DG) [(a) and (b)], lonidamine (LND) [(c) and (d)], oxythiamine (OT) [(e) and (f)], and 4-(chloromethyl) benzoylchloride (4-CMBC) [(g) and (h)]. Significant changes ($p<0.05$) marked with stars were highlighted in the graphs.

We note that other authors also observed a decrease in the concentration of free NAD(P)H after treatment with 2-DG and a concomitant increase of the ${\mathrm{NAD}(\mathrm{P})}^{+}/\mathrm{NAD}(\mathrm{P})\mathrm{H}$ ratio in the cytosol.^4,4748.^–49 In our study, an extremely low concentration of 2-DG was used ($25\mu \mathrm{M}$) compared to previous works; however, significantly decreased levels of free:bound NAD(P)H ratios and increased average fluorescence lifetimes could be detected. Irrespective of the mechanism underlying these variations,²⁷ we show that FLIM microscopy provides quantitative data with sufficient precision to be an extremely sensitive method to detect subtle fluctuations in the metabolism of living cells. We are unaware of other studies having previously demonstrated this aspect of multiphoton FLIM microscopy.

We have been able to measure the changes in the relative contributions of free to protein-bound NAD(P)H before and after the treatment over the entire cellular volume, including its nuclear, cytosolic, and mitochondrial pools, where we observed a significant decrease after treatment for 1 and 2 h and a small decrease after 48 h in cells cultured with serum. However, the full picture is somewhat more nuanced. For example, the ratio of free:bound NAD(P)H (${\alpha }_1/{\alpha }_2$) in the cytosol and the nucleus are slightly higher than that in the mitochondria.¹⁴ 2-DG appears to affect the glycolysis in the cytosol, causing a significant decrease of the (${\alpha }_1/{\alpha }_2$) ratio, reflecting a decrease of the ratio of free-overbound NAD(P)H. Because free NAD(P)H can diffuse through the nuclear pore, the change of free NAD(P)H in the nuclear compartment may be associated with the decrease of free NAD(P)H in the cytosol, or with its consumption there. NADH cannot simply pass into the mitochondria because the inner mitochondrial membrane is impermeable to NADH and ${\mathrm{NAD}}^{+}$. Thus, the (${\alpha }_1/{\alpha }_2$) ratio in the mitochondria is almost unchanged before and after our treatment, indicating that 2-DG may have little effect on the mitochondrial NADH equilibrium (data not shown here).

We also noticed a consistent difference in NAD(P)H florescence lifetimes of cells grown with and without serum for all experiments, also reported previously.²² The addition of serum to the culture media is known to increase the growth rate of the cells by increasing the glucose metabolism in response to signaling.^50,51 Serum starvation dramatically increases the fraction of bound NAD(P)H in the nucleus.⁵² The addition of a small amount of serum (2%) causes a decrease in the quantity of nuclear free NAD(P)H, which influences the overall colocalization of free and bound NAD(P)H within the nucleus, and an increase in free NAD(P)H within the cytoplasm. The cells exposed to 10% serum show a larger fraction of free NAD(P)H in the nucleus.⁵² This effect of serum was also evident in our experiments in the control groups, where the (${\alpha }_1/{\alpha }_2$) ratios were always lower in the presence of serum as compared to serum-free samples.

3.3.

Very Small Metabolic Changes Induced by LND Can be Detected Using NAD(P)H Fluorescence Lifetime

We treated MCF10A cells with just $20\mu \mathrm{M}$ of LND in the presence and the absence of serum, at a lower concentration than that used in earlier works (45 to $600\mu \mathrm{M}$).^1,36 The data extracted from FLIM images are presented in Figs. 2(c) and 2(d). For all measured samples, the lifetimes for free and bound NAD(P)H were in the following ranges: ${\tau }_1$ in 0.38 to $0.49\pm 0.03\mathrm{ns}$, ${\tau }_2$ in 1.6 to $2.3\pm 0.05\mathrm{ns}$, respectively, with free:bound NAD(P)H ratios in the range of 1.7 to $2.5\pm 0.03$ and average lifetimes ${\tau }_{\mathrm{m}}$ in 0.8 to $1.05\pm 0.05\mathrm{ns}$ range. The mean fluorescence lifetimes ${\tau }_{\mathrm{m}}$ decreased after the treatment, contrary to the trends observed for the 2-DG treatment [see Fig. 2(c)]. A significant decrease ($p<0.05$) was observed in the mean fluorescence lifetime after 24 h of treatment with LND in the presence and absence of serum. LND also significantly increased the ratio of free:bound NAD(P)H after 24 h for cells grown without serum. The same trend was observed for cells with serum, but it was not significant. These results are noteworthy since the change in intracellular pH induced by $80\mu \mathrm{M}$ LND inhibition of lactate transport was only marginal.¹ However, Figs. 2(c) and 2(d) detected clearly observable differences in both NAD(P)H lifetime and the ratio of free:bound NAD(P)H after 24 h.

3.4.

Effect of Oxythiamine on the NAD(P)H Fluorescence Lifetime

FLIM images for MCF10A cells treated with $20\mu \mathrm{M}$ OT exhibited much less variability compared to samples treated with other inhibitors, as apparent in the lifetime data presented in Figs. 2(e) and 2(f). At this concentration, marked effects on cells (motility) have been observed.³⁵ For all measured samples, lifetimes for free and bound NAD(P)H were in the following ranges: ${\tau }_1$ in 0.39 to $0.56\pm 0.005\mathrm{ns}$, ${\tau }_2$ in 2.25 to $2.65\pm 0.005\mathrm{ns}$, respectively, with free:bound NAD(P)H ratios in the range of 1.8 to $2.5\pm 0.05$ and average lifetimes ${\tau }_{\mathrm{m}}$ in 1.1 to $1.2\pm 0.05\mathrm{ns}$ range. The mean fluorescence lifetime ${\tau }_{\mathrm{m}}$ of NAD(P)H in MCF10A cells treated with OT in the presence or absence of serum are presented in Fig. 2(e). No significant differences were detected between control and treatment groups in the presence and in the absence of serum. The relative contributions of free and bound NAD(P)H [Fig. 2(f)] show only very small changes in the presence of serum, with a significant decrease ($p<0.05$) only for samples treated for 48 h, and in absence of serum, the only significant difference was observed within the first hour of treatment.

In our study, there was no significant change in the mean fluorescence lifetime ${\tau }_{\mathrm{m}}$ of NAD(P)H after the treatment with OT [Fig. 2(e)]. NADPH is produced by PPP and has the same fluorescence properties as NADH. Thus, alteration of NADPH production by OT may have contributed to the difference in the experiment ratios of free:bound NAD(P)H. As the concentration of NADPH is about four to seven times lower than NADH in a range of tissue types,⁵³ the effect of metabolic perturbations on NAD(P)H fluorescence should be lower by a similar factor. Taken together, this suggests that the decrease in free NAD(P)H observed after 1 and 48 h of OT treatment [Fig. 2(f)] was caused by the inhibitory effect of OT. However, we cannot exclude the possibility that other metabolic pathways contributed to the result.

3.5.

Effect of 4-(Chloromethyl) Benzoyl Chloride on the NAD(P)H Fluorescence Lifetime

The lifetime data extracted from FLIM images of MCF10A cells treated with $40\mu \mathrm{M}$ 4-CMBC in the presence or absence of 3% serum are presented in Figs. 2(g) and 2(h). For all measured samples, the lifetimes for free and bound NAD(P)H were in the following ranges: ${\tau }_1$ in 0.44 to $0.56\pm 0.005\mathrm{ns}$, ${\tau }_2$ in 2.0 to $2.5\pm 0.05\mathrm{ns}$, respectively, with free:bound NAD(P)H ratios in the range of 1.6 to $2.6\pm 0.05$ and average lifetimes ${\tau }_{\mathrm{m}}$ in 0.90 to $1.20\pm 0.005\mathrm{ns}$ range. Significant increases in the average lifetime of NAD(P)H fluorescence were observed at 2 and 24 h in the absence of serum, which were associated with cell death by 48 h, whereas a significant decrease in fluorescence lifetime was observed in the presence of serum at 24 h.

Figure 2(h) depicts the relative contributions of free and bound NAD(P)H after the treatment with 4-CMBC. A significant decrease ($p<0.05$) in the ${\alpha }_1/{\alpha }_2$ ratio was observed in the presence of serum after 1, 2, and 48 h of treatment, while the 24-h sample showed a small increase compared to its control, well correlating with the changes in an average lifetime.

Regardless of its mechanism of action, in our experiment, 4-CMBC caused a slightly higher change in the mean fluorescence lifetime of NAD(P)H in the absence of serum and the changes were correlated with changes in free:bound NAD(P)H ratios between treated and control samples. This result may be attributed to a significant decrease in the viability of MCF10A cells grown in medium without serum treated with 4-CMBC, as the cell count was reduced to $\sim 78\%$ compared to controls after 24 h and $<10\%$ after 48 h (in the latter case, cells were not suitable for imaging). Cell viability was not altered in the presence of serum, indicating some protective activity against the inhibitor.

3.6.

Overview of the Effect of Investigated Inhibitors on Contribution of Free NAD(P)H

In order to obtain an improved overview of the changes in the previously presented data, we calculated the relative percentage difference between the contribution of free NAD(P)H measured for MCF10A cells treated with different inhibitors and their respective nontreated controls for cells grown in the two types of medium. They are shown in Fig. 3 for samples grown without serum [Fig. 3(a)] and with 3% of serum [Fig. 3(b)]. The differences in contribution of free NAD(P)H for all cells treated with different inhibitors are within $\pm 30\%$. In the absence of serum, the variability is higher than for samples treated in the presence of 3% serum, and the highest reduction in free NAD(P)H was observed for treatment with 2-DG and 4-CMBC for 2 h, at 25 and 19%, respectively. OT exhibited the least effect for MCF10A treated cells with or without serum. In contrast, LND induced the highest increase in free NAD(P)H contribution after 24 h treatment for samples without and with serum, 29 and 15%, respectively.

Fig. 3

Percentage difference in contribution of short component of lifetime, assigned to free NAD(P)H as compared to respective control and normalized to that control, for MCF10A cells treated with different inhibitors and measured for samples and controls incubated for 1, 2, 24, and 48 h from inhibitor treatment. Control samples were MCF10A cells cultured for the same times but without inhibitors. The experiments were conducted with medium without serum—graph (a) and with 3% serum—graph (b) and following inhibitors: 2DG, LND, OT, and 4-CMBC (see legend in the graphs).

For samples with 3% serum, the changes in free NAD(P)H were less pronounced. For all treated samples, a reduction in free NAD(P)H was observed in the first hour for all inhibitors, after which cells showed some recovery for the next 2- and 24-h periods, followed by a decrease for samples treated for 48 h. Based upon our results, the strongest inhibition of free NAD(P)H (which can be associated with decreased metabolic activity) was observed for inhibitors in the following order: $4\text{-}\mathrm{CMBC}>2\text{-}\mathrm{DG}>\mathrm{OT}$, and LND exhibited the apparent opposite effect. These LND-induced changes in the contribution of free NAD(P)H presumably involve changes in intracellular pH. LND is a well-established inhibitor of monocarboxylate transport,^1,36 so that lactate secretion should be impaired by LND, leading to cytoplasmic accumulation of lactate, which should decrease the cytoplasmic pH. We propose the following rationalization for our observed results. Mild LND-induced cytoplasmic acidification should induce a stress response and accompanying activation of glycolysis to produce pyruvate and, subsequently, lactate. Since the $20\mu \mathrm{M}$ concentration of LND employed was lower than the $50\mu \mathrm{M}$ required to achieve 50% inhibition of lactate secretion,⁵⁴ lactate should still have accumulated in the extracellular medium, which would have diminished the magnitude of any cytoplasmic pH change attributable to LND inhibition of lactate transport. However, alternative mechanisms could not be ruled out.

3.7.

Effects of Inhibitors on Lactate Secretion

Figure 4 shows results of lactate secretion measurements for MCF10 cells grown in medium with 3% serum, and Fig. 5 presents results of the same experiment for cells without serum, treated with four inhibitors at 1, 2, 24, and 48 h. Here, we measured the level of L-lactate concentration in the supernatant of treated cells. These results show that for cells grown in medium with serum only, the treatment with 2-DG led to a noticeable decrease in lactate secretion at 1 and 2 h. For other inhibitors, the changes in the level of L-lactate upon inhibitor treatments were insignificant. A small decrease of concentration was observed at 1- and 48-h treatments with OT and in the first hour for 4-CMBC. LND-treated MCF10A cells grown in medium with serum showed a slight increase in the level of lactate after 1 and 2 h after the inhibitor treatment and a slight decrease for the 24-h sample. In the case of 4-CMBC, a small increase was observed only for the 2 h of treatment, but for other periods, no differences were detected.

Fig. 4

Results of lactate production for MCF10 cells grown in medium with 3% serum, treated with various inhibitors: (a) 2DG, (b) LND, (c) OT, (d) 4-CMBC, and measured at intervals of 1, 2, 24, and 48 h from inhibitor treatment. Bars correspond to control samples—shown in white, treated samples—shown as dashed bars. L-lactate concentration in supernatant taken from controls and treated samples was measured by absorbance measurements and compared to calibration curve obtained using standards from the Cayman glycolysis kit.

Fig. 5

Results of glycolysis measurements for MCF10 cells grown in medium without serum, treated with various inhibitors: (a) 2DG, (b) LD, (c) OT, and (d) 4-CMBC, and measured at intervals of 1, 2, 24, and 48 h from inhibitor treatment. All data are plotted using the same conventions as in Fig. 4.

In cells placed in a medium without serum, slightly higher variations between control and treated cells were observed and are shown in Fig. 5. The treatment with 2-DG inhibitor led to a noticeable decrease in secreted lactate for 1 and 2 h after treatment. An increase in lactate concentration in cells treated with LND and with OT was observed after 2 h of treatment. For cells treated with 4-CMBC, no differences were observed with respect to controls, and only a small decrease for the 24-h treatment.

In all lactate secretion experiments performed using the Cayman glycolysis kit, the concentration levels of lactate are measured after secretion to the media. Levels were in the 0.1- to $1\text{-}\mu \mathrm{M}$ range after 1 and 2 h of inhibitor treatment and in 1- to $10\text{-}\mu \mathrm{M}$ range for 24 and 48 h. This difference in measured concentrations over time is related to increased number of secreting cells. These experiments detected very little change in lactate secretion as a result of the application of the inhibitors. The small differences observed here for different inhibitors are consistent with the results of FLIM analysis and with the low levels of inhibitors employed here. We confirmed that the secretion of L-lactate was most affected by 2-DG (which caused a decrease in lactate concentration) and LND (which caused an increase). These results are consistent with our hypothesis that the low LND concentration used here partially inhibits lactate transport, inducing mild cytoplasmic acidification, which in turn hypothetically increases glycolytic activity. The poorest consistency between FLIM and measured lactate secretion levels was observed for the 4-CMBC inhibitor, as this inhibitor caused a significant decrease in cell viability. In that respect, in FLIM measurements, the data were collected from regions of similar cell density for treated and untreated cells and should accurately correspond to internal free:bound NAD(P)H ratio differences. However, the lactate secretion experiment measures bulk cell population properties as it indirectly quantifies bulk lactate secreted to the surrounding medium by all cells in the culture. Decreased cell viability upon treatment made comparisons at the later time points impossible to complete.

3.8.

Effect of Mitochondria Membrane Potential Inhibition on NAD(P)H Lifetime

Figures 6(a) and 6(b) show the results of NAD(P)H fluorescence lifetime for cells incubated with and without serum and treated with CCCP for different times. It shows that the ratio of free:bound NAD(P)H decreases in cells treated with CCCP and incubated with or without serum, with larger fluctuations in the absence of serum. There is also more variation in the NAD(P)H life time in cells treated with CCCP incubated with medium than that without serum. Figure 6(c) shows the ratio of red to green fluorescence, which is an indicator for mitochondria membrane potential in cells stained with JC-1, a sensitive marker for mitochondrial membrane potential that exhibits a green to red fluorescence emission shift upon its potential-dependent accumulation in mitochondria.⁵⁵ Figures 6(d) and 6(e) show confocal microscopy images of control cells imaged in the green and red channels, while Figs. 6(f) and 6(g) show cells treated with CCCP for 1 h, both grown in medium with 3% serum. The proportional increase in green fluorescence of JC-1 after CCCP treatment indicates mitochondrial depolarization by CCCP.

Fig. 6

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibitor on metabolic activity of MCF10 cells measured [(a) and (b)] by lifetime measurements and (c) by staining using JC-1 membrane potential stain as a function of time from inhibitor treatment. Results shown are for the cells grown in medium with 3% serum: graph (a) and black curve in (c), and for cells grown in medium without serum: graph (b) and red curve in (c). In graph (a) and (b), average lifetime is shown as dashed bar with values corresponding to left axis and free:bound NAD(P)H ratios (ratio of contributions of short-lifetime component to long-lifetime component in measured lifetime) as red line with values corresponding to right axis. Cell images are shown for control [(d) and (e)] and for sample after 60 min from CCCP treatment [(f) and (g)], both grown in medium with 3% serum. Green images were acquired at excitation/emission of $540\mathrm{nm}/590\mathrm{nm}$ and red images at $570\mathrm{nm}/610\mathrm{nm}$.

The pH of the mitochondrial matrix (${\mathrm{pH}}_{\mathrm{mit}}$) is higher than the cytosolic pH (${\mathrm{pH}}_{\mathrm{cyt}}$). Since the addition of CCCP leads to a complete dissipation of the pH gradient over the mitochondrial inner membrane,⁵⁶ a rapid acidification of ${\mathrm{pH}}_{\mathrm{mit}}$ is expected.⁵⁷ This has an effect on free NAD(P)H fluorescence, where the average lifetime of free NAD(P)H marginally decreases with increasing pH,⁵⁸ but is greatly affected by pH-induced structural changes to the proteins with which it interacts⁵⁹ and, therefore, the ratio of free:bound NAD(P)H. CCCP also affects the intracellular environment through the deregulation of the ion balance of the cell by the translocation of protons through membranes. Thus, the pH of the cytoplasm inside the cell equilibrates with the pH of the medium outside, which is higher than the reported intracellular pH for both ${\mathrm{pH}}_{\mathrm{mit}}$ and ${\mathrm{pH}}_{\mathrm{cyt}}$.⁶⁰ Therefore, the average lifetime of both free and bound NAD(P)H is affected by changes of pH. In our system, reduction of NAD(P)H lifetime and changes in the ratio of free:bound NAD(P)H were observed only in the absence of serum (Fig. 6). We presume that serum induces undefined intracellular signaling activity that can mitigate the effects of CCCP, presumably by sustaining proton gradients. However, that conclusion remains tentative.