4.1. Electrophoresis conditions and DNA damage levels

We have previously studied experimentally [17] the conditions which determine the level of migration measured in the comet assay. Cell cultures of two types were treated with genotoxicants and then assayed for DNA damage using different physical conditions during electrophoresis. Linear regression of the experimental data in [17] shows highly linear relationships for V/cm and time (Table 1) and an inverse relationship for agarose. Fitting straight lines to the figures in [17] results in the regression data (for human blood mononuclear cells) in Table 1.

There was no effect of altering the electrophoresis current (between 210 and 400 mA), except for a slight downward trend which is explained as an indirect effect on the electric potential [17].

#### 4.2. Position- and time-dependent variations in local electric potential

Since the local electric potential determines the mobility of DNA fragments in the comet assay, it is essential that the electric potential is defined at all positions of the electrophoresis platform. This may be particularly important if the comet gels are small in the surface. We have described a high-throughput comet assay with 96 minigels, each of 4 μl, placed in an array of Gelbond plastic membranes as substrate [18, 19]. The minigels give results which are highly similar to results obtained with standard glass slides. We also evaluated this revised comet assay with respect to variation in DNA damage levels measured within one electrphoresis. Based on preliminary studies [18], DNA damage measured in parallel samples depended significantly on the circulation of the liquid during electrophoresis. We now report on a series of measurements of local electric potentials, with the purpose of identifying possible timedependent differences in electrophoresis between neighboring samples. A multi-electrode gauge was made (Figure 1) consisting of 20 evenly spaced (5 mm) platinum electrodes, each covered with a thin plastic tube except for 1 mm protruding free end.

This multi-electrode gauge was placed in an electrophoresis tank with the free electrode ends immersed in a thin layer of agarose of the same concentration (0.675%) as used to embed cells in the comet assay (Figure 2) [20]. The electrode ends were a fraction of a millimeter above the


Table 1. Linear regression of data from [17], with goodness-of-fit. The regression curves for electric potential (V/cm) and time (min) were both forced through zero.

Figure 1. Multi-electrode gauge connected to a multiplexing digital voltmeter (Agilent 34972A with Multiplexer 34901A) allowing the potential at up to 20 electrodes to be sequentially scanned at intervals.

Figure 2. A schematic diagram illustrating the electrophoresis tank (1) with the platform (2) and the electrodes (3). During electrophoresis the tank was filled with liquid (4) and an electrode gauge (5) was connected to an electrode plate (6) and placed across the center of the platform with its electrodes covered in agarose (7) when performing the measurements of the electric potentials.

GelBond films. The dimensions of the tank are listed in Table 2. The electric potential of each minielectrode could be scanned and recorded automatically during 1 s, for every 10 s, using a multiplexer and a digital voltmeter with input resistance in the mega-ohm range. This also allowed continuous measurements of the temperature in the solution.

Systematic studies of the role of circulation were carried out, using external circulation at 21, 58, 109, 201, and 285 ml/min. With a total volume of 1640 ml, this compares to 1, 4, 7, 12, and 17% exchange of volume per min, respectively. Apparently, there were significant differences in electrode potentials at neighboring positions and also time-dependent changes (Figures 3 and 4, upper panels). With circulation, however, these variations were much reduced (Figures 3 and 4, lower panels; figures show graphs for 109 ml/min circulation). In Figure 4, the data from Figure 3 have been converted into local electric potentials.

The calculated total migration of DNA in each electrophoresis is derived from the timeintegrated electric potential (dimension V/cm\*min). Figure 5 presents these data for electrophoresis during 25 min, with and without circulation. Corrections due to some differences in

Figure 3. Measured voltage across the platform during electrophoresis. The relative voltage at each electrode and temperature over time during an electrophoresis time of 25 mins from one representative experiment with (109 ml/min) and without circulation. The number of each electrode and the temperature are indicated to the right of the figure. The blue thick lines in both plots represent the temperature. The figures are from one representative experiment [20].

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These experiments were repeated several times; data averaged for all electrodes are presented in Figure 6. In total, there was a consistent and clearly reduced inter-experimental variation,

The electric potential measurements were paralleled by analysis of DNA damage in cell samples from a batch of human blood mononuclear cells exposed to a fixed dose of ionizing radiation (X-rays) on ice. This dose (8 Gy) was established from a dose-response curve (Figure 7). Materials

4.3. Position-dependent variations in DNA damage measured in samples exposed to

the spacing between the electrodes were included in the calculations.

when circulation was used (109 ml/min).

irradiation


Table 2. Physical dimensions of the electrophoresis tank and the electrode plate.

Figure 3. Measured voltage across the platform during electrophoresis. The relative voltage at each electrode and temperature over time during an electrophoresis time of 25 mins from one representative experiment with (109 ml/min) and without circulation. The number of each electrode and the temperature are indicated to the right of the figure. The blue thick lines in both plots represent the temperature. The figures are from one representative experiment [20].

GelBond films. The dimensions of the tank are listed in Table 2. The electric potential of each minielectrode could be scanned and recorded automatically during 1 s, for every 10 s, using a multiplexer and a digital voltmeter with input resistance in the mega-ohm range. This also

Figure 2. A schematic diagram illustrating the electrophoresis tank (1) with the platform (2) and the electrodes (3). During electrophoresis the tank was filled with liquid (4) and an electrode gauge (5) was connected to an electrode plate (6) and placed across the center of the platform with its electrodes covered in agarose (7) when performing the measurements of

Figure 1. Multi-electrode gauge connected to a multiplexing digital voltmeter (Agilent 34972A with Multiplexer 34901A)

Systematic studies of the role of circulation were carried out, using external circulation at 21, 58, 109, 201, and 285 ml/min. With a total volume of 1640 ml, this compares to 1, 4, 7, 12, and 17% exchange of volume per min, respectively. Apparently, there were significant differences in electrode potentials at neighboring positions and also time-dependent changes (Figures 3 and 4, upper panels). With circulation, however, these variations were much reduced (Figures 3 and 4, lower panels; figures show graphs for 109 ml/min circulation). In Figure 4, the data from Figure 3

Description Length (mm) Width (mm) Height (mm)

Tank 290 262 70 Platform 180 262 26 Side wells 2 110 262 26 Electrode plate 180 262 9

Table 2. Physical dimensions of the electrophoresis tank and the electrode plate.

allowed continuous measurements of the temperature in the solution.

allowing the potential at up to 20 electrodes to be sequentially scanned at intervals.

68 Electrophoresis - Life Sciences Practical Applications

have been converted into local electric potentials.

the electric potentials.

The calculated total migration of DNA in each electrophoresis is derived from the timeintegrated electric potential (dimension V/cm\*min). Figure 5 presents these data for electrophoresis during 25 min, with and without circulation. Corrections due to some differences in the spacing between the electrodes were included in the calculations.

These experiments were repeated several times; data averaged for all electrodes are presented in Figure 6. In total, there was a consistent and clearly reduced inter-experimental variation, when circulation was used (109 ml/min).

### 4.3. Position-dependent variations in DNA damage measured in samples exposed to irradiation

The electric potential measurements were paralleled by analysis of DNA damage in cell samples from a batch of human blood mononuclear cells exposed to a fixed dose of ionizing radiation (X-rays) on ice. This dose (8 Gy) was established from a dose-response curve (Figure 7). Materials

Figure 4. Stabilization of the voltage at each electrode position with circulation. Corrected voltage variations per cm (calculated from Figure 3) during the electrophoresis time of 25 mins for each of the electrodes, with 109 ml/min or without circulation (Figure from [20]). One representative experiment. The number of each electrode is indicated to the right of the figure. In this specific experiment, electrode #16 showed particularly large variations without circulation, whereas other electrodes were affected in other experiments.

and methods used in the experiments were the same as in [18], based on our system of 96 minigels on GelBond films. For scoring of comets, a fully automated system (Imstar PathfinderTM, Paris, France) was used, for two reasons; (1) unsupervised scoring avoids errors introduced by operator interactions; (2) there were very large numbers of samples to be scored (384 samples per electrophoresis). To ensure that automated scoring gives comet results comparable to the standard semi-automated scoring (Comet IV, Perceptive Instruments Ltd., Bury St Edmunds, UK) we used both systems to generate the data in Figure 7; they gave highly similar rates of median DNA damage (denoted as Tail%DNA, i.e. the fluorescence of the comet tail divided by the total fluorescence of the comet), versus the dose of radiation. At 15 Gy the Tail%DNA is saturated and the dose response is no longer linear; this data was therefore omitted in the linear regression analysis. The dose 8 Gy which was used in cell exposures is in the linear part of the curve and is well below saturation.

circulation, and differences between samples belonging to different rows or columns in the

Figure 5. Voltage gradient at each electrode position during electrophoresis with circulation at different flow rates. The corrected electric potentials were time-integrated during an electrophoresis time of 25 mins, and are presented (V/cm) during one (average) mins. Data are from three experiments without circulation or with circulation at 21 ml/min (upper panel); the blue, green, and red bars correspond to each of the three experiments. For higher flow rates (58, 109, 201, and

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The experiments included electrophoresis of either one film placed in the center of the plat-

The median (Tail%DNA) of all scored comets in each sample was used as the basis for further statistical analyses. In some calculations, the values were weighted, based on the number of comets scored in each sample. Normality of distribution of DNA damage in each cell sample was tested using a Lilliefors test, in MATLAB R2014b (significance level 5%). Differences between groups (rates of circulation, sample positions in rows and columns) were tested with one-way ANOVA. A structure of statistics obtained from the one-way ANOVA was used to perform the multiple comparison tests, which determined whether any group mean was significantly different; two group means were significantly different if their intervals were disjoint. A Wilcoxon

array (8 columns 12 rows) on each of four films in one electrophoresis.

285 ml/min), the data (lower panels) represent one representative experiment. Figure from [20].

form, or 4 films totaling 384 samples covering the whole platform [18] (Figure 8).

A large number of comet electrophoresis experiments was carried out with irradiated (8 Gy) mononuclear cells. We studied differences in means between experiments, with and without

Figure 5. Voltage gradient at each electrode position during electrophoresis with circulation at different flow rates. The corrected electric potentials were time-integrated during an electrophoresis time of 25 mins, and are presented (V/cm) during one (average) mins. Data are from three experiments without circulation or with circulation at 21 ml/min (upper panel); the blue, green, and red bars correspond to each of the three experiments. For higher flow rates (58, 109, 201, and 285 ml/min), the data (lower panels) represent one representative experiment. Figure from [20].

circulation, and differences between samples belonging to different rows or columns in the array (8 columns 12 rows) on each of four films in one electrophoresis.

and methods used in the experiments were the same as in [18], based on our system of 96 minigels on GelBond films. For scoring of comets, a fully automated system (Imstar PathfinderTM, Paris, France) was used, for two reasons; (1) unsupervised scoring avoids errors introduced by operator interactions; (2) there were very large numbers of samples to be scored (384 samples per electrophoresis). To ensure that automated scoring gives comet results comparable to the standard semi-automated scoring (Comet IV, Perceptive Instruments Ltd., Bury St Edmunds, UK) we used both systems to generate the data in Figure 7; they gave highly similar rates of median DNA damage (denoted as Tail%DNA, i.e. the fluorescence of the comet tail divided by the total fluorescence of the comet), versus the dose of radiation. At 15 Gy the Tail%DNA is saturated and the dose response is no longer linear; this data was therefore omitted in the linear regression analysis. The dose 8 Gy which was used in cell exposures is in the linear part of the curve and is

Figure 4. Stabilization of the voltage at each electrode position with circulation. Corrected voltage variations per cm (calculated from Figure 3) during the electrophoresis time of 25 mins for each of the electrodes, with 109 ml/min or without circulation (Figure from [20]). One representative experiment. The number of each electrode is indicated to the right of the figure. In this specific experiment, electrode #16 showed particularly large variations without circulation,

A large number of comet electrophoresis experiments was carried out with irradiated (8 Gy) mononuclear cells. We studied differences in means between experiments, with and without

well below saturation.

whereas other electrodes were affected in other experiments.

70 Electrophoresis - Life Sciences Practical Applications

The experiments included electrophoresis of either one film placed in the center of the platform, or 4 films totaling 384 samples covering the whole platform [18] (Figure 8).

The median (Tail%DNA) of all scored comets in each sample was used as the basis for further statistical analyses. In some calculations, the values were weighted, based on the number of comets scored in each sample. Normality of distribution of DNA damage in each cell sample was tested using a Lilliefors test, in MATLAB R2014b (significance level 5%). Differences between groups (rates of circulation, sample positions in rows and columns) were tested with one-way ANOVA. A structure of statistics obtained from the one-way ANOVA was used to perform the multiple comparison tests, which determined whether any group mean was significantly different; two group means were significantly different if their intervals were disjoint. A Wilcoxon

Figure 6. Average electric potentials measured in several experiments. Time-integrated corrected voltage per cm averaged for all electrodes during an electrophoresis time of 25 mins for each measurement with (109 ml/min, lower panel) and without (upper panel) circulation. The standard deviations are shown as red error bars and illustrate the degree of variation in voltage per cm for each measurement. Figure from [20].

Figure 7. Dose-response curves showing DNA damage versus radiation dose. One experiment; scoring of DNA damage in 96 samples with the semi-automated (Perceptives) and the automated scoring (IMSTAR Pathfinder) systems. Irradiation with X-rays (0–15 Gy) and circulation (109 ml/min) during electrophoresis. The Tail%DNA relative to the head is given as the median value of at least 50 comets in each sample. Linear regression lines are shown, with linear relations y = 3.99 + 4.37x and y = 0.1 + 4.30x, and coefficients of variation (CV) R<sup>2</sup> = 0.72 and R<sup>2</sup> = 0.96, for IMSTAR and Perceptives,

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Figure 8. Illustration of positioning of Gelbond films with one film (left; 96 minigels), or four films (right; 384 minigels) on

respectively. The experimental points at dose 15 Gy were omitted from the linear regression.

a platform in an electrophoresis tank. Figure from [20].

rank sum test—not requiring the normal distribution of samples—was used when the Tail% DNA values were shown not to be normally distributed by the Lilliefors test.

We observed small differences in Tail%DNA levels in experiments using different rates of circulation. This is illustrated in Table 3 and in Figure 9, both based on weighted data (to avoid that low numbers of scored comets should be given too much weight).
