Determination of the effects of additional NaCl input in cell cultivation.

<p>Metabolic activity of both NIH 3T3 (<b>A</b>, <b>B</b>) and SH-SY5Y cells (<b>C</b>, <b>D</b>) grown in culture medium containing 6.4 (Pos.Ctrl., reference), 6.26, 6.12 and 5.93 mg/ml NaCl corresponding to 1:10, 1:5 and 1:3 dilution of the NaCl stock in culture medium was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 13–23; SH-SY5Y, n = 24–27). The resulting formazan dye intensities were also related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SE_M. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, *p ≤ 0.05).</p

Microscopic characterization of the morphology of SH-SY5Y cells following exposure to varying NaCl concentrations.

<p>SH-SY5Y cells were cultivated either in normal cell culture medium containing 6.4 mg/ml NaCl as reference (<b>A</b>) or in culture medium containing 6.26 mg/ml (1:10) (<b>B</b>), 6.12 mg/ml (1:5) (<b>C</b>) and 5.93 mg/ml (1:3) NaCl (<b>D</b>). Microscopic images demonstrated reduced cell attachment and growth in a concentration dependent manner without any signs of cytotoxicity.</p

Ultrastructural morphology of NIH 3T3 cells following exposure to Pt-Diss.

<p>After cultivation either without Pt as reference (<b>A</b>) or in culture medium containing 6.0 μg/ml (<b>B</b>), 0.11 μg/ml (<b>C</b>) or 0.02 μg/ml Pt (<b>D</b>) the ultrastructure of 3T3 fibroblasts was compared. They demonstrated mitochondrial swelling (arrow in <b>B</b>) only at the highest tested Pt concentration. At 0.11 μg/ml Pt the cells showed greater phagocytic activity (arrowhead in <b>C</b>). Lower amount of Pt in the culture medium induced no morphological changes in comparison with the control. Size of bars: 2 μm.</p

Microscopic characterization of the morphology of NIH 3T3 cells following exposure to Pt-NP.

<p>NIH 3T3 cells were cultivated either without any additional Pt particles as reference (<b>A</b>) or in culture medium containing 25 μg/ml (<b>B</b>), 50 μg/ml (<b>C</b>) and 100 μg/ml (<b>D</b>) of the Pt-NP. The images demonstrated highly uniform cell adhesion without any morphological impairment throughout the cell cultures assays with varying Pt-NP concentrations. Size of bars: 200 μm.</p

Determination of the effects of Pt-Diss with varying Pt-Diss concentrations in cell cultivation.

<p>Metabolic activity of both NIH 3T3 (<b>A</b>) and SH-SY5Y cells (<b>B</b>) grown in culture medium supplied with 0.82 μg/ml– 8.2 μg/ml Pt-Diss concentration was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–16; SH-SY5Y, n = 10). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SE_M. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).</p

Determination of the effects of Pt-NP with varying concentrations in cell cultivation.

<p>Metabolic activity of both NIH 3T3 (<b>A</b>) and SH-SY5Y cells (<b>B</b>) grown in culture medium supplied with 5 μg/ml– 100 μg/ml Pt-NP was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–14; SH-SY5Y, n = 11–15). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SE_M. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).</p

Microscopic characterization of the morphology of NIH 3T3 cells following exposure to Pt-Diss.

<p>NIH 3T3 cells were cultivated either without any additional Pt-Diss as reference (<b>A</b>) or in culture medium containing 8.2 μg/ml (<b>B</b>), 4.1 μg/ml (<b>C</b>), 1.64 μg/ml (<b>D</b>) and 0.82 μg/ml (<b>E</b>) of the Pt components. Microscopic images demonstrated emerging cytotoxic effects of Pt-Diss in a concentration-dependent manner between 1.64 μg/ml and 8.2 μg/ml Pt-Diss concentration. Beyond Pt-Diss concentration of 1.64 μg/ml cell adhesion and morphology appeared normal, whereas a Pt-Diss concentration of 8.2 μg/ml strongly reduced NIH 3T3 cell growth and probably induced cell death. Size of bars: 200 μm.</p

Ultrastructure of the SH-SY5Y cell line cultivated in absence and presence of Pt-Diss.

<p>After cultivation either without any Pt as reference (<b>A</b>) or in culture medium containing 6.0 μg/ml (<b>B</b>), 0.11 μg/ml (<b>C</b>) or 0.02 μg/ml Pt (<b>D</b>) SH-SY5Y were characterized by large euchromatic nucleus, abundant endoplasmic reticulum and a few synaptic granules (arrowheads). At the highest tested Pt concentration these cells were adversely affected, as proven by mitochondrial swelling (arrow in <b>B</b>). A smaller amount of Pt in the culture medium induced no morphological changes in comparison with the control. Size of bars: 2 μm.</p

Microscopic characterization of the morphology of SH-SY5Y cells following exposure to Pt-NP.

<p>SH-SY5Y cells were cultivated either without any additional Pt particles as reference (<b>A</b>) or in culture medium containing 25 μg/ml (<b>B</b>), 50 μg/ml (<b>C</b>) and 100 μg/ml (<b>D</b>) of the Pt-NP. Morphology and adhesion behavior of the SH-SY5Y cells did not change throughout the cultivation assays at varying Pt-NP concentrations. Size of bars: 200 μm.</p

Microscopic characterization of the morphology of SH-SY5Y cells following exposure to Pt-Diss.

<p>SH-SY5Y cells were cultivated either without any additional Pt-Diss as reference (<b>A</b>) or in culture medium containing 8.2 μg/ml (<b>B</b>), 4.1 μg/ml (<b>C</b>), 1.64 μg/ml (<b>D</b>) and 0.82 μg/ml (<b>E</b>) of the Pt components. Microscopic images demonstrated emerging cytotoxic effects of Pt in a concentration dependent-manner between 1.64 μg/ml and 8.2 μg/ml Pt-Diss concentration. Cell adhesion and growth appeared stable following exposure to Pt-Diss concentration of around 1.64 μg/ml. Even spontanous neurite sprouting could be observed, whereas the 8.2 μg/ml of the Pt-Diss concentration strongly induced detachment of the SH-SY5Y cells and subsequent cell death. Size of bars: 150 μm.</p