An electrochemical system for the study of trans plasma membrane electron transport in whole eukaryotic cells

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Supporting Information

An Electrochemical System for the Study of Trans-Plasma Membrane Electron Transport

in Whole Eukaryotic Cells

Harry G. Sherman

, Carolyn Jovanovic

, Snow Stolnik

, Frankie J.Rawson

*

†Divisions of Regenerative Medicine and Cellular Therapies, School of Pharmacy, University of Nottingham, NG7 2RD, UK

∞Division of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, NG7 2RD, UK

‡ Walgreens Boots Alliance, Nottingham, NG2 3AA, UK

Contents:

Method S-1. Stability of FIC in cell culture conditions.

Figure S-1. Simultaneous quantification of iron redox states for the stability of FIC and FOC at 37ºC and 5% CO2.

Method S-2. Investigation of electrode fouling.

Method S-3. Bicinchoninic acid assay.

Figure S-2: Iron redox state concentration calibration study.

Figure S-3. Calibration curve for potassium ferrocyanide (FOC) and potassium ferricyanide (FIC).

Figure S-4. Investigation of electrode fouling.

Method S-4 Relating to Figure 3A. Assessment of growth rate using a Tecan plate reader with cell counting and viability functions.

Figure S-5 Relating to Figure 3A. Growth rate study for Calu-3 cells.

Figure S-6. Toxicity of phosphate buffered saline (PBS) and Hanks’ Balanced Salt Solution (HBSS).

Method S-5. pH testing of Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC), before and after cell incubation.

Figure S-7. pH testing of Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC), before and after cell incubation.

Method S-6. Inductively coupled plasma mass spectrometry (ICP-MS).

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Method S-1. Stability of FIC in cell culture conditions. 0.01 mM FIC and FOC (Acros Organics) solutions were both made in Hanks’ Balanced Salt Solution (HBSS). The two solutions were mixed in a 1:1 ratio to give a 0.01 mM solution containing 0.005 mM FIC and 0.005 mM FOC. The resulting solution was subject to linear sweep

voltammetry, as de-scribed above, with and without incubation for 2 hours at 37°C, 5% CO2. All parameters and procedures for electro-chemical analysis were as outlined for the section on calibration procedure. HBSS was processed in the same way as tested samples and values obtained subtracted for normalisation purpose.

Figure S-1. Simultaneous quantification of iron redox states for the stability of FIC and FOC at 37ºC and 5% CO2.

Figure S-1. Mean linear sweep voltammogram obtained for solutions of ferricyanide/ferrocyanide (FIC/FOC) at a

1:1 ratio at a final concentration of 0.01 mM with Hanks’ Balanced Salt Solution (HBSS) buffer as the supporting electrolyte. Solutions were incubated at 37˚C in 5% CO2 atmosphere for 2 hours (red) and no incubation at room

temperature and atmospheric conditions for 2 hours (black). Linear sweep voltammetric analysis was performed at a starting potential of 500 mV and an end potential of -150 mV, at a scan rate of 10 mV s-1. Pseudo-steady state anodic current values were used to calculate FOC concentration (Right panel, crossed bar), and pseudo-steady state cathodic values used to calculate FIC concentration (Right panel, white bars). Total iron concentration (Right panel, whole bar) was calculated by adding FOC and FIC together. The right panel depicts total iron concentration for before and after incubation at 37˚C in 5% CO2, each total concentration bar also demonstrates the amount of FIC

and FOC for before and after incubation, represented as parts of the total iron concentration. Paired t-test indicated no significant difference between scans before and after incubation, p= 0.363. Error bars show ± 1SD from the mean. N=3, n=3.

The stability of FIC and FOC at our defined cell culture conditions, over two hours, needed to be determined. This was done to ensure that both redox states of iron did not precipitate out of solution, or degrade, in the conditions tested, which would alter our total iron concentration. This involved testing a non-incubated and incubated sample of a 1:1 mixture of FIC (0.005 mM) and FOC (0.005 mM). This experiment also demonstrates our ability to simultaneously detect two iron redox states, a major advantage of our application. Figure 1 demonstrates that there is no change in the current values between curves in the voltammogram when our iron mixture is subjected to heat and increased CO2, in line with the conditions during cell experiments. The equations from Figure 2 were used to

calculate concentrations of FIC (bottom equation) and FOC (top equation), and these concentrations added to determine our total iron concentration. Paired two-tailed t-test showed no significance between the total iron concentrations calculated at p = 0.289, meaning that our data indicates that there is no iron degradation or precipitation when subjected to the conditions tested. We can see our total iron concentration is also near 10 µM, which indicates our system is valid.

Method S-2. Investigation of electrode fouling. 0.01 mM FIC (Acros Organics) solution was made in Phosphate

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Method S-3. Bicinchoninic acid assay. Following removal of the supernatant for electrochemical analysis of cell-conditioned samples, each well was washed three times for 5 minutes each with PBS. 1 ml of 2% triton X-100 solution was then added to each well and the plates incubated at 37°C for 20 minutes. Following this the lysed cell-triton samples were transferred to a microcentrifuge tube and centrifuged at 16000g for 20 minutes. The supernatant was removed and transferred to a fresh microcentrifuge tube before analysis using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific Ltd) and the manufactures recommended protocol.

Figure S-2 Voltammograms of 0.01 mM FIC calibration curve.

Figure S-2. Iron redox state calibration study. 0.01 mM potassium ferricyanide (FIC) and ferrocyanide (FOC) made up in HBSS were mixed in ratios 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 1:9 and 0:10. Each mixture was subject to linear sweep voltammetry between 500 mV and -150 mV at 37˚C. Scan rate, 10 mV.s-1. A clear upward shift is observed in the voltammogram with increasing FOC concentration. N=3, n=3.

Figure S-3.Calibration curve for potassium ferrocyanide (FOC) and potassium ferricyanide (FIC).

Figure S-3. Calibration curve for potassium ferrocyanide (FOC) and potassium ferricyanide (FIC) for

concentrations between 0 and 10 µM, with 95% prediction bands shown (dotted lines). FOC and FIC were made in Hank’s Balanced Salt Solution (HBSS). Linear sweep voltammetric analysis was performed at a starting potential of 500 mV and an end potential of -150 mV, at a scan rate of 10 mV s-1, and at 37˚C. GraphPad Prism 7.01 software was used to calculate linear regression lines in addition to 95% prediction bands, which are expected to encompass 95% of future data points. SD error bars are also shown for all points. N=3, n=3.

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Figure S-4. Mean linear sweep voltammogram obtained for a 0.01 mM solution of ferricyanide (FIC) in Phosphate Buffer Saline (PBS), tested before and after the microelectrode had been used with cell-incubated material. Linear sweep voltammetric analysis was performed at a starting potential of 600 mV and an end potential of -250 mV, at a scan rate of 10 mV s-1, 37 ˚C. Pseudo-steady state anodic current values were used to calculate FOC concentration and pseudo-steady state cathodic values used to calculate FIC concentration. Total iron concentration was calculated by adding FOC and FIC together. The FIC solution was tested before (Right panel, dotted bar) and after (right panel, crossed bar) the electrode has been used with cell-incubated material. Paired t-test indicated no significant difference between scans before and after cell-related electrode use. (p=0.1406). Error bars show ± 1SD from the mean. N=9, n=3.

It was important to determine whether the electrode was likely to be fouled in salt based buffer when working with cell solutions. This involved testing a 0.01 mM FIC solution before and after the electrode was used with cell-incubated buffer. We found there to be no change in the concentration of iron before and after testing, and therefore conclude that there was no element of electrode fouling present. This is interesting as it addresses one of the potential reasons for the reduction in total iron concentration that we see in our cell-induced iron reduction experiments.

Method S-4 Relating to Figure 3A. Assessment of growth rate using a Tecan plate reader with cell counting and

viability functions.

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Figure S-5 Relating to Figure 1. Growth rate study for Calu-3 cells.

Figure S-5. Growth rate study for Calu-3 cells. Calu-3 cells were seeded at 150’000 cells/well in a 12 well plate and grown for 8 days. Viability was tested by tryphan blue exclusion and viable cell number counted using a Tecan microplate reader (Tecan Ltd, Weymouth, UK). 150’000 cells/well seeding density yielded multiple growth sections, with a central plateau phase. Seeding density was increased to yield three distinct growth phases (Figure 3). N=1, n=3.

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Figure S-6. (A) Lactate dehydrogenase assay for determination of buffer type and cellular membrane integrity, over 2h. Two-way ANOVA with Sidak's multiple comparisons test used for statistical analysis. P= 0.230, 0.003 and 0.011 for Calu-3, H1299 and A549 Phosphate Biffer Saline-Hank’s Balanced Salt Solution (PBS-HBSS)

comparisons, respectively. N=3, n=3. (B) MTS assay data for cellular metabolic activity when incubated with FIC in HBSS for 2h. Relative metabolic profiles for selected cells with Dulbecco’s Modified Eagle Medium (DMEM) as negative control. Cells show a decline in relative metabolic rate after doses of 10 mM FIC. SEM error bars shown. N=3, n=3.

LDH assay data is shown in Figure S-6A. Dulbecco’s Modified Eagle Medium (DMEM) was selected as the negative control to mimic ideal conditions, and assigned 0% LDH release. Triton X-100, a well-used detergent, was used as positive control to represent 100% cell death and therefore maximum signal, it was assigned 100% LDH release. For both A549 and H1299 cells HBSS caused a lower rate of membrane perturbation at significance of p = 0.011 and p = 0.003, respectively. Calu-3 cells showed no preference for PBS or HBSS at p = 0.230. As a result of these experiments HBSS was chosen as the supporting electrolyte. The rationale behind this decision was that if PBS was causing a higher incidence of membrane perturbation there would likely be a higher incidence of intracellular species that were electrochemically active released into the supporting electrolyte. In addition, this would be more likely to cause electrode fouling. Thus, choosing HBSS would ensure there was less electrochemical interference with the electrochemical system, and there would also be a lower incidence of cell death. It was important to establish iron was having no effect on cell viability.

Having selected our supporting electrolyte, FIC ability to induce cytotoxicity on each cell line was investigated using the MTS assay. The other reason for choosing this assay was to provide values for the mitochondrial

metabolic rate of our cell lines. This would allow us to test the hypothesis that tPMET systems are used in cells with high metabolic rates as a means to aid in the regeneration of NAD+ and facilitate increased rates of glycolysis3. We wanted to investigate this as the electron transport chain (ETC) within the mitochondria is involved with NAD+ regeneration by feeding electrons from NADH into the ETC. DMEM was selected as negative control to mimic ideal conditions, and assigned 100% cell viability. Triton X-100 was used as positive control to represent 100% cell death, and assigned 0% viability. A HBSS control was also set up to ensure we could test the metabolic rate effects of HBSS against ideal conditions using DMEM. As can be seen in FigureS-6B there is no effect upon relative metabolic rate until a concentration of 10 mM FIC is reached. As a result of this investigation a concentration of 0.01 Mm FIC was selected for subsequent experiments. This ensured the chosen concentration was well within the non-cytotoxic range, but also meant a higher signal-to-noise ratio was more likely, as any reduced iron would constitute a larger portion of the overall iron concentration if the total concentration was smaller. Interesting results were achieved when comparing HBSS relative metabolic activities to DMEM controls (100%). For Calu-3 cells there was no cytotoxic effect at p = 0.696, whereas H1299 and A549 cells were affected at p = 0.0002 and p = 0.002, respectively. This provides an insight into what is causing cytotoxic effects in our system. For Calu-3 cells it appears to be inherent viability deficit even in ideal conditions, whereas H1299 and A549 appear to be viable in ideal conditions and undergo stress when transferred into an alternate medium.

By taking the absorbance values in DMEM conditions for all cell types, and normalising using the number of cells from our growth study we could gain some information about the cells mitochondrial metabolic rate. Interestingly, Calu-3 cells had the highest normalised absorbance (and hence mitochondrial metabolic rate) of 9.56 x 10-5A.U. per cell, whilst A549 cells were similar at 9.23 x 10-5 A.U. per cell, and H1229 significantly different to both at 7.16 x 10-5 A.U. per cell (p = 0.0001 for A549/Calu-3 cells vs H1299 cells).

Method S-5. pH testing of Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC), before

and after cell incubation. Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC) in HBSS were

measured using a pH meter (model 3510, Jenway) before and after two hours incubation with Calu-3, H1299 or A549 cells, at 37˚C in 5% CO2. Cells were plated and grown as described for cellular electrochemistry experiments.

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Figure S-7. pH testing of Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC), before and after cell incubation.

Figure S-7. Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC) in HBSS were measured using

a pH meter (model 3510, Jenway) before and after two hours incubation with Calu-3, H1299 or A549 cells, at 37˚C in 5% CO2. Statistics were determines using two-way ANOVA with Sidak’s multiple comparison test. Error bars

show ± 1SEM from the mean. N=3, n=3.

It was important to look at the pH changes when incubating with cells as there were noticeable changes in the voltammograms when using cells to reduce iron. There is a pH change for HBSS buffer (with or without FIC) when incubated with all cell lines at p < 0.0001. For HBSS the pH when non-incubated is 7.62, which drops to 7.17, 6.95 and 6.95 for Calu-3, H1299 and A549 respectively. This is a small change for both HBSS of 0.45 pH for Calu-3 cells and 0.67 pH for both H1299 and A549. For FIC the pH when non-incubated is 7.62, which drops to 7.13, 7.11 and 7.05 for Calu-3, H1299 and A549 cells respectively. This is again a small change of 0.49, 0.51 and 0.57 respectively. These small changes that we see may be enough to slightly alter the conditions at the electrode surface and thus cause the ‘flattening’ of the voltammogram from the altered half wave potential. There is a difference in pH for some cell lines when comparing between HBSS and FIC incubated with the same cells for two hours. For HBSS and FIC only (no incubation) there was no change at p >0.9999. For Calu-3 cells there was no significant difference at 0.7806, for H1299 there was a difference at p = 0.0003, and also for A549 at p = 0.0310. This also could contribute to changes in our voltammograms as the baseline subtraction may not be completely aligned to the sample. However, this does not affect our results as by using the first derivative method of assessing the pseudo-steady state we are able to accurately pinpoint the relevant voltages for steady states despite changes to the half-wave potential or voltammogram shape.

Method S-6. Inductively coupled plasma mass spectrometry (ICP-MS).

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Figure S-8. Inductively coupled plasma mass spectrometry (ICP-MS).

Figure

Figure S-1. Simultaneous quantification of iron redox states for the stability of FIC and FOC at 37ºC and 5%  CO 2

Figure S-1.

Simultaneous quantification of iron redox states for the stability of FIC and FOC at 37ºC and 5% CO 2 p.2
Figure S-2. Iron redox state calibration study. 0.01 mM potassium ferricyanide (FIC) and ferrocyanide (FOC) made

Figure S-2.

Iron redox state calibration study. 0.01 mM potassium ferricyanide (FIC) and ferrocyanide (FOC) made p.3
Figure S-2 Voltammograms of 0.01 mM FIC calibration curve.

Figure S-2

Voltammograms of 0.01 mM FIC calibration curve. p.3
Figure S-4. Mean linear sweep voltammogram obtained for a 0.01 mM solution of ferricyanide (FIC) in Phosphate

Figure S-4.

Mean linear sweep voltammogram obtained for a 0.01 mM solution of ferricyanide (FIC) in Phosphate p.4
Figure S-5. Growth rate study for Calu-3 cells. Calu-3 cells were seeded at 150’000 cells/well in a 12 well plate and

Figure S-5.

Growth rate study for Calu-3 cells. Calu-3 cells were seeded at 150’000 cells/well in a 12 well plate and p.5
Figure S-5 Relating to Figure 1. Growth rate study for Calu-3 cells.

Figure S-5

Relating to Figure 1. Growth rate study for Calu-3 cells. p.5
Figure S-7. pH testing of Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC), before and  after cell incubation

Figure S-7.

pH testing of Hanks’ Balanced Salt Solution (HBSS) and 0.01 mM Ferricyanide (FIC), before and after cell incubation p.7
Figure S-8.  Inductively coupled plasma mass spectrometry (ICP-MS).

Figure S-8.

Inductively coupled plasma mass spectrometry (ICP-MS). p.8

References