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Establishment of a cell micropatterning method for the quantitative assessment of the organization of the keratin filament network

3.5 Distribution of KIFs in normalized cells
Since in most cases only a small number of normalized cells existed the distribution of human keratin 5 filaments in HaCaT B10 cells and human keratin 13 filaments in AK13-1 cells was assessed based on individual cell images. Independent of the pattern shape and size both keratin filament networks formed a dense network braiding the nucleus and radiated toward the cell periphery. Compared to the compact perinuclear network a decreasing toward the corners was observed, particularly evident in teardrop-shaped (Fig. 13 A“), triangle-shaped (Fig. 13 C“, 15 B‘ and 15 C‘), square-shaped (Fig. 14 D‘) and arrow-shaped (Fig. 14 C‘) cells.

Fig. 13 Individual examples of HaCaT B10 cells spread on 700 µm² micropatterns. HaCaT B10 cells were seeded on a CYTOOchip™ and allowed to spread on the micropatterns for 24 h before fixation. Micropatterns are detected by fluorescently labeled FN (A, B and C), nuclei by DAPI staining (superposed on contrast image [TL] in A', B' and C') and human keratin 5 through EYFP signal (A'', B'' and C''). A merged image of fluorescently labeled FN, DAPI and human keratin 5-EGFP is shown in A''', B''' and C'''. Bar, 10 µm.

Fig. 13 Individual examples of HaCaT B10 cells spread on 700 µm² micropatterns. HaCaT B10 cells were seeded on a CYTOOchip™ and allowed to spread on the micropatterns for 24 h before fixation. Micropatterns are detected by fluorescently labeled FN (A, B and C), nuclei by DAPI staining (superposed on contrast image [TL] in A‘, B‘ and C‘) and human keratin 5 through EYFP signal (A“, B“ and C“). A merged image of fluorescently labeled FN, DAPI and human keratin 5-EGFP is shown in A“‘, B“‘ and C“‘. Bar, 10 µm.

Fig. 14 AK13-1 on printed PDMS substrates. AK13-1 cells were seeded on printed PDMS substrates and allowed to spread on micropatterns with an average cell spreading area of 1100 µm² for 24 h before fixation. Micropatterns are detected through secondary antibodies mixed in the FN solution (A, B, C and D), nuclei by DAPI staining (superposed on human keratin 13 EGFP-signal in A', B', C' and D'). A merged image of micropatterns, DAPI and human keratin 13-EGFP is shown in A'', B'', C'' and D''. Bar, 10 µm.

Fig. 14 AK13-1 on printed PDMS substrates. AK13-1 cells were seeded on printed PDMS substrates and allowed to spread on micropatterns with an average cell spreading area of 1100 µm² for 24 h before fixation. Micropatterns are detected through secondary antibodies mixed in the FN solution (A, B, C and D), nuclei by DAPI staining (superposed on human keratin 13 EGFP-signal in A‘, B‘, C‘ and D‘). A merged image of micropatterns, DAPI and human keratin 13-EGFP is shown in A“, B“, C“ and D“. Bar, 10 µm.

Fig. 15 AK13-1 on printed PDMS substrates. AK13-1 cells were seeded on printed PDMS substrates and allowed to spread on micropatterns with an average cell spreading area of 1100 µm² for 24 h before fixation. Micropatterns are detected through secondary antibodies mixed in the FN solution (A, B and C), nuclei by DAPI staining (superposed on human keratin 13 EGFP-signal in A', B' and C). A merged image of micropatterns, DAPI and human keratin 13-EGFP is shown in A'', B'' and C''. Bar, 10 µm.

Fig. 15 AK13-1 on printed PDMS substrates. AK13-1 cells were seeded on printed PDMS substrates and allowed to spread on micropatterns with an average cell spreading area of 1100 µm² for 24 h before fixation. Micropatterns are detected through secondary antibodies mixed in the FN solution (A, B and C), nuclei by DAPI staining (superposed on human keratin 13 EGFP-signal in A‘, B‘ and C). A merged image of micropatterns, DAPI and human keratin 13-EGFP is shown in A“, B“ and C“. Bar, 10 µm.

While the average distribution of human keratin 5 in HaCaT B10 cells could quantitatively assessed by generating heat maps of cells spread on crossbow-shaped (Fig. 16 A), disc-shaped (Fig. 16 B) and Y-shaped (Fig. 16C) micropatterns with an average cell spreading area of 700 µm², the average distribution of human keratin 13 in AK13-1 cells was assessed by generating heat maps of cells spread on teardrop-shaped (Fig. 16 E), disc-shaped (Fig. 16 F), arrow-shaped (Fig. 16 G) and square-shaped (Fig. 16 H) micropatterns with an average cell spreading area of 1100 µm².
Images were exported into ImageJ and analyzed with CYTOO tooL for Image Processing-Reference cell, an ImageJ macro provided by CYTOO Inc. Images were aligned, stacked, averaged and pseudo-colored to represent regions of high and low intensity where blue hues represent low frequency of protein localization and red hues represent high frequency of protein localization. If necessary, the macro was adjusted for individual image stacks so that it could identify the corresponding fluorescent micropatterns in order to delineate regions centered on micropatterns and to realign the image stack in all channels.

Fig. 16 Heap maps illustrating the average distribution of keratin intermediate filaments. A-C) Average distribution of human keratin 5 in HaCaT B 10 cells spread on 700 µm² micropatterns. E-H) Average distribution of human keratin 13 in AK13-1 cells spread on 1100 µm² micropatterns. Calibration bar shows quantitative protein density of the KIFs mapped in linear pseudo-colored panel that spans the full range of intensity within each image, blue hues representing low intensity and red hue representing high intensity. n, number of cells used to generate the heat map. Bar, 10 µm

Fig. 16 Heap maps illustrating the average distribution of keratin intermediate filaments. A-C) Average distribution of human keratin 5 in HaCaT B 10 cells spread on 700 µm² micropatterns. E-H) Average distribution of human keratin 13 in AK13-1 cells spread on 1100 µm² micropatterns. Calibration bar shows quantitative protein density of the KIFs mapped in linear pseudo-colored panel that spans the full range of intensity within each image, blue hues representing low intensity and red hue representing high intensity. n, number of cells used to generate the heat map. Bar, 10 µm

The observation of the average distribution of human keratin 5 in HaCaT B10 cells and of human keratin 13 in AK13-1 cells as seen in individual cell images (Fig. 13-15) could be confirmed for the corresponding shapes. A high average distribution of the KIFs could be observed around the nuclei together with a decrease of the average distribution of the KIFs toward the cell periphery.

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