AKLIDES® Nuk Software

Evaluation and Results

The AKLIDES® Nuk is an adaption of AKLIDES® Kyto for detection of double-strand breaks in cell preparations. The experience with characterization of immunological fluorescence pattern facilitates the valid recording and detection of punctuate fluorescence (foci), which become visible due to specific double strand-break markers.

This time, measurements are carried out by a 60x lens on object slides. An additional contrasting staining in the blue DAPI channel ensures a robust focussing despite various monolayer cell preparations. Subsequent morphological description allows for a directed selection of cellular phases for the detection of strand-breaks (e.g. exclusion of metaphases). Pre-defined cell-line dependent profiles (e.g. average nucleus size) make for a fast interpretation without the need of manually adjusting measurement parameters. Artefact structures are therefore detected and excluded from further analysis.

Highly sensitive measurement of the γ-H2AX signal in the green FITC channel ensues along several z-planes (cell-line dependent). This makes it possible to detect every strand-break independent of the focal plane. The used algorithms produce a 3-dimensional array composed of the detected x,y and z coordinates of the foci. Succeeding filtrations facilitate the perimeter of for example highly prominent or weakly marked strand-breaks. For every single focus, detailed parameters as size, intensity, brightness, shape and position are gathered.
The statistical evaluation of measurement values by, for example, robust median calculation of single cell measurements and the definition of minimum requirements for the quality of foci (visible in at least 2 planes) permit a valid calculation of the measured parameters.



Results

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