The comparison of BrdU, IdU and CldU affinities for the anti-bromodeoxyuridine antibody clone B44.

<p>The analysis of the signal in cells incubated for 1 hour with BrdU or IdU or CldU or in the control, non-labelled, cells. The copper(I) ions and exonuclease III was used for BrdU/CldU/IdU revelation. The values are normalised for the IdU signal equal to 1. The data are presented as SB/B ± SEM (signal with the background/background).</p

Scheme of the system for the antibody analysis.

<p>The scheme of the system for the antibody analysis is shown. The signal intensities are normalised to the signal obtained with the highest antibody concentration equal to 100%.</p

The effect of BrdU detection protocol on the affinity of the anti-bromodeoxyuridine clones to BrdU.

<p>The analysis of the signal in the cells after a 1-hour incubation with BrdU or in the control, BrdU non-labelled, cells is shown. BrdU was revealed with 2N HCl, 4N HCl, DNase I or copper(I) ions with exonuclease III. The signal intensity is plotted as the SB/B ratio ± SEM. (signal with the background/background).</p

MTT assay results.

<p>The results of the MTT assay are shown. The impact of BrdU, IdU and CldU on HeLa and HCT116 cells is shown. The data represents the mean ± SEM.</p

The EC50 values (A) and fingerprints (B) of the affinity of the anti-bromodeoxyuridine antibodies.

<p>(A) The EC50 values for three oligonucleotide probes containing BrdU at the 3' or 5' end or in the central part of the oligonucleotide chain ± SEM are shown for each antibody. In the last column, the mean of these three EC50 values ± SEM is shown. The values marked with a star were estimated and probably are below the real values. (B) The fingerprint involving the EC50 values for the oligonucleotides containing BrdU at the 3' or 5' end or in the central part of the oligonucleotide chain are shown in a bar graph. The affinity of the clones is expressed as 1/EC50 ± SEM in percent. The data are normalized to the signal obtained for the oligonucleotide with BrdU at the 5' end. This signal is equal to 100%.</p

The bivariate analysis of the IdU signal in HeLa cells.

<p>The bivariate analysis of the replication signal in HeLa cells after a 20-minute incubation with 10 mM IdU (A) and in control, non-labelled HeLa cells (B) is shown. The copper(I) ions and exonuclease III was used for IdU revelation in DNA. The percent of cells in G1 phase was ~50%, in S phase ~36% and in G2/M phase ~14%.</p

Affinity constants (A) and the scatter plot (B) of tested antibody clones.

<p>(A) The affinity constants for anti-bromodeoxyuridine antibody clones and oligonucleotides containing BrdU at the 3' or 5' end or in the central part of the oligonucleotide chain are shown. The values marked with a star were estimated and probably are above the real values. The data are presented as the mean ± SEM. (B) The scatter plot shows the distribution of the anti-bromodeoxyuridine clones with respect to the mean and variability of the EC50 values calculated from the EC50 values for oligonucleotides containing BrdU at the 3′ or 5′ end or in the central part of the oligonucleotide chain. The variability is expressed as the ratio between the standard deviation and the mean of the EC50 values and is plotted against the EC50 mean.</p

The effect of various treatments on the DNA content (A) and DNA histogram (B).

<p>(A) The DAPI signal in cell nuclei after the treatment with 2N HCl, 4N HCl, DNase I, copper(I) ions with exonuclease III and in the control cells is shown. DAPI signal of the control non- treated cells is equal to 100%. The data are normalized to % of the signal of control cells. The data are shown as the mean nuclear signal ± SEM. (B) The histograms of nuclear integral DAPI signal in non-treated cells (control) or cells treated with 2N HCl, 4N HCl, copper(I) ions and exonuclease III or DNase I is shown.</p

The cleavage of plasmid DNA using copper(II) sulfate and sodium ascorbate in presence of oxygen.

<p><b>A</b>) The results of the cleavage of the plasmid DNA are shown. The plasmid DNA (pEXP5-NT/CALM3) was incubated in an aqueous solution of copper(II) sulfate and sodium ascorbate in the presence of oxygen for 0, 1, 5, 10, 20, 30 and 60 minutes (lines 1, 2, 3, 4, 5, 6 and 7, respectively). Line M represents the DNA molecular mass marker (GeneRuler™ DNA ladder Mix, Fermentas). The lowering of integral intensity toward 60 minutes apparently resulted from the increase of the low-weight DNA that escaped the gel. <b>B</b>) The centers of density of the bands from the above-mentioned experiments (<b>a</b>) plotted against the time of the incubation are shown.</p

The increase of azido dye concentration and acid treatment results in a non-specific signal.

<p>The picture shows examples of the detection of incorporated EdU in DNA by means of a click reaction with 0.02 mM fluorescent dye without the HCl treatment (images labelled as control) and of detection with 0.2 mM fluorescent azido dyes and the antibody clone BU1/75 (Cy3 anti-rat and FITC anti-rat secondary antibodies) in cells treated with 4N HCl. Note that the higher concentration of fluorescent azido dyes and the subsequent treatment with 4N HCl led to a non-specific signal mainly in the nucleolus area. Barr: 20 µm.</p