Table 1.
Primary and secondary antibodies for immunostaining of cell-specific antigens in the SGC.
Fig 1.
Representative microscopic view of the morphology of the HEI-OC1 cells following exposure to Na2(PtCl6).
HEI-OC1 cells were cultivated either without Na2(PtCl6) as an experimental control (A) or in culture media supplemented with 8 ng/μl (B), 10 ng/μl (C), 12 ng/μl (D), 14 ng/μl (E) or 16 ng/μl of Na2(PtCl6) (F). HEI-OC1 cells incubated with 15% DMSO for 1.5 h served as a cell death control (G). Normal cell growth without any morphological anomalies could be observed in the experimental control, whereas cell culture assays exposed to Na2(PtCl6) resulted in a concentration-dependent decrease in cell density and adhesion as well as loss of cell contacts. Microscopic images shown are representative of a total of N = 10 experiments. Each experiment was performed in triplicate (n = 3). Scale bars: 100 μm.
Fig 2.
Representative live cell staining of HEI-OC1 cells for evaluation of cell death in presence of Na2(PtCl6).
Calcein AM (green fluorescence, stained in false colour yellow in the images) and EthD (red fluorescence, stained in false colour magenta in the images) were used for fluorescent live cell staining following Na2(PtCl6) exposure. The plasma membrane-permeable Calcein AM represents vital following its cleavage to calcein in the cytoplasm and conversion to the green fluorescent dye in the presence of Ca2+ ions. In contrast, EthD is only able to infiltrate cells with leaky membranes to intercalate double-stranded DNA emitting red fluorescent light. HEI-OC1 cells cultivated without Na2(PtCl6) represented the experimental control group showing any cell loss (A), whereas those exposed to 8 ng/μl (B), 10 ng/μl (C), 12 ng/μl (D), 14 ng/μl (E) and 16 ng/μl (F) Na2(PtCl6) demonstrated dose-dependent cell death induction, as indicated by an increase in red fluorescent dots and a decrease in vital cells monitored by Calcein AM triggered green fluorescence. Similarly, DMSO-induced cytotoxicity in the HEI-OC1 cells could be shown by red fluorescent EthD-DNA complexes as a result of membrane disintegration (G). The images of live cell staining of the HEI-OC1 cells are representative of a total of N = 3 experiments. Each experiment was performed in triplicate (n = 3). Scale bars: 200 μm.
Fig 3.
Presence of mitophagy in HEI-OC1 cells following exposure to Na2(PtCl6).
The normal ultrastructure of the HEI-OC1 cells also remained unchanged in the presence of lower doses of Na2(PtCl6) (8 ng/μl Na2(PtCl6)) (A). Following incubation with 14 ng/μl Na2(PtCl6) (B), only some HEI-OC1 cells survived, whereas the other cells underwent necroptosis characterized by incomplete cell membranes and organelles as well as the loss of cytoplasmic components. At higher magnification of single surviving cells mitochondria could be seen in an increased amount, but were partly seen inside autophagolysosomes (C, arrowheads). The multilamellar vacuoles in the surroundings demonstrated the remnants of the digested mitochondria. Scale bars: 2 μm.
Fig 4.
Determination of the oxidative activity of HEI-OC1 cells following exposure to Na2(PtCl6).
The oxidative activity of HEI-OC1 cells exposed to varying concentrations of Na2(PtCl6) was examined by indirect reduction of resazurin in the presence of dehydrogenases to the highly fluorescent resorufin. The resulting fluorescence intensities, measured as fluorescence units (FU) after 48 h of cultivation, were relative to those of the untreated control group and calculated as percentages [%]. Each data point is presented as the mean ± SEM. Repeated measures ANOVA with Tukey’s multiple comparison test was performed for statistical assessment (*p ≤ 0.05). The dashed line represents the cytotoxicity limit (70%). A total of N = 10 independent experiments were conducted. Each experiment was performed in triplicate (n = 3).
Fig 5.
Determination of the survival rate and neurite outgrowth of the SGN following exposure to Na2(PtCl6).
Relative quantification of the survival rate (A) and measurement of the neurite extensions (B) of the SGN cultivated in completed neuromedium supplied with 15–35 ng/μl Na2(PtCl6). Anti-neurofilament staining was used to visualize the soma and nerve fibers of the SGN. Each data point is presented as the mean ± SEM of the (A) percentage of labeled soma (N = 5, n = 3 of each Na2(PtCl6) concentration) relative to the control group without Na2(PtCl6) treatment and (B) length of neurites (N = 5, n = 15 neurons of each Na2(PtCl6) concentration). Repeated measures ANOVA with Tukey’s multiple comparison test was performed for statistical assessment. According to ISO 10993–5:2009, the dashed line in (A) represents the cytotoxicity limit (70%) [22].
Fig 6.
Representative immunofluorescence images of non-neuronal cells in SGC culture assays supplied with Na2(PtCl6).
DAPI was used for nucleic staining (A, E, L, M, Q, U). Vim (labeled fibroblasts and glial cells, green, stained yellow in the images) and p75 antigen (binding to glial cells, red, stained magenta in the images) staining was found in the control group without Na2(PtCl6) incubation, indicating vital cells (A-D), whereas administration of 20 ng/μl (I-L), 25 ng/μl (M-P), 30 ng/μl (Q-T) and 35 ng/μl (U-X) resulted in detachment of the cells from the cell culture dish and retraction of the cell-cell contacts. SGC cultivation assays exposed to 2.5% DMSO served as a cell death control (E-H). The immunofluorescence images of the non-neuronal cells are representative of a total of N = 3 independent experiments. Each experiment was performed in triplicate (n = 3). Scale bars: 100 μm.
Fig 7.
Representative fluorescence images of OCex hair cells following organotypic cultivation in the presence of Na2(PtCl6).
Confocal laser scanning microscopy images of the soma of hair cells without any Na2(PtCl6) treatment showed intact rows of the IHC and OHC in all cochlear parts (A-C). In contrast, the application of 25 ng/μl (D-F), 35 ng/μl (G-I) and 45 ng/μl (J-L) Na2(PtCl6) resulted in a dose-dependent decrease in the number of IHC in all cochlear turns, followed by the decrease in the number of OHC at the highest Na2(PtCl6) concentration used in this study (J-L). Magenta crosses indicate the loss of hair cells. The fluorescence images of the OCex hair cells are representative of a total of N = 3 independent experiments. Each experiment was performed in triplicate (n = 3). Scale bar for all images: 20 μm.