Concentration of hydrogen peroxide in 60 s plasma-treated DMEM.

<p>Note the continuous decrease of hydrogen peroxide over various time periods. (n = 7, Mann-Whitney <i>U</i>-test, ***p<0.001).</p

Argon plasma treatment and medium temperature.

<p>The average temperature in DMEM during plasma treatment up to 120 s is 25°C and remains constant up to a plasma treatment time of 180 s. Thus, negative thermal effects in the plasma-treated cell culture medium DMEM are excluded.</p

Cell membrane morphology of mHepR1 cells.

<p><i>a)</i> The surface structure of cells after 24 h in normal, untreated medium (left column) presents elongated microvilli with high densities, which extend over the entire cell surface. In contrast, the mHepR1 cells in 60 s plasma-treated medium (right column) show extremely shortened microvilli accompanied by a reduced density (bar = 4 µm, 5,000× magnification, SEM DSM 960 A, Carl Zeiss). <i>b)</i> The significantly shortened microvilli are impressively visible using a higher magnification (bar = 2 µm, 15,000× magnification, FE-SEM Zeiss Merlin VP compact, Carl Zeiss) (p<0.001, student's <i>t-</i>test, n = 410–500 microvilli). <i>c)</i> Scheme: microvilli at the cell surface in normal epithelial cells (left) and cells exposed to plasma-treated DMEM (right).</p

Oxygen content of 60 s plasma-treated DMEM.

<p>Note that in the plasma-treated complete DMEM the molecular oxygen concentration is decreased by half, which is equalised after only one day. (PreSens oxygen sensor, n = 3, student's <i>t</i>-test, *p<0.05, ** p<0.01, ***p<0.001).</p

Online-monitoring of respiration of living mHepR1 epithelial cells.

<p>Note that the respiration is immediately disturbed after addition of complete DMEM plasma-treated for 60 s (arrow) which was stored for 4 days at 37°C. (Bionas 1500 analysing system, n = 3, student's <i>t</i>-test * p<0.05 for all values from the time point of 3.5 hours).</p

Tight junction architecture in the mHepR1 epithelial cell monolayer.

<p><i>a)</i> Note the continuous ZO-1 bands between mHepR1 cells in normal, untreated cell medium DMEM (left column). In contrast, 60 s plasma-treated DMEM stored for 1 d induces large openings between cell margins (arrow, right above), indicating a break of tight junctions. Plasma-treated DMEM (60 s) stored for 7 d induces an irregular cytoplasmic distribution of the ZO-1 proteins (arrow, right below). (AxioObserver.Z1, Carl Zeiss, bars = 5, 10 µm). <i>b)</i> The plasma treatment time of 120 s of the DMEM and storage of 7 d induces not only a retraction of the ZO-1 protein into the cytoplasm but also a nearly complete loss of the cell-cell contacts (arrow), while normal cells show a strong bonding (arrowhead). Note the long-lasting argon plasma effect of cell medium on structural organisation of cells. (AxioObserver.Z1, bar = 50 µm, the windows indicate the areas of the magnified view below).</p

Online-monitoring of cell adhesion of living mHepR1 cells.

<p>Note that complete DMEM plasma-treated for 60 s, which was stored for 4 days at 37°C, induced the cells to detach from the sensor chip indicating the loss of adhesion capacity. The arrow indicates the time point of medium addition. (Bionas 1500 analysing system, n = 3, student's <i>t</i>-test * p<0.05 for all values from the time point of 4 hours).</p

Workflow.

<p>Cell biological investigations after cultivation with argon plasma-treated, long-time stored cell medium DMEM.</p

Representative gel filtration chromatogram resulting from: (a) application of untreated complete medium, (b) complete argon-gas treated medium, (c) complete medium with 60 s or (d) 120 s of plasma treatment.

<p>The arrowheads mark the one peak which alters considerably after plasma treatment. (n = 6 independent experiments).</p

Oxygen content of cell culture medium immediately after plasma treatment for 60 and 120 s. (PreSens oxygen sensor, mean ± SD, n = 3; student's <i>t</i>-test).

<p>*p<0.05 <i>vs.</i> untreated,</p><p>***p<0.001 <i>vs.</i> untreated.</p><p>Oxygen content of cell culture medium immediately after plasma treatment for 60 and 120 s. (PreSens oxygen sensor, mean ± SD, n = 3; student's <i>t</i>-test).</p