Cell viability (WST-1 assay) after 4 h of ZnO exposure observed in a mutant Jurkat T-cell line deficient in caspase-9 (JMR) versus their caspase-9-restored counterpart (JMR/C9).

<p>Data of treated cells are related to corresponding control cells (100% activity, n = 3).</p

Effects of ZnO exposure in murine bone marrow-derived macrophages of p47^phox−/− versus wt animals.

<p>(A) Superoxide detection via lucigenin-amplified chemiluminescence depicted in arbitrary units (AU). (B) FACS analysis of sideward scatter related granularity to investigate particle uptake (n = 2). (C) Cell viability determined by WST-1 assay (n = 2). (D) Content of hypodiploid cells determined by FACS analysis after 7-AAD staining (n = 2). STS: staurosporine.</p

Fluorescent staining of RAW 264.7 macrophages after 4 h treatment with ZnO or staurosporine (STS) using TUNEL assay.

<p>Representative multichannel images are shown for apoptotic DNA fragmentation (green staining) and corresponding nuclei (blue staining). Original magnification 100×.</p

Investigation of the role of ZnO solubilization on the effects in RAW 264.7 cells.

<p>(A) FACS analysis of RAW 264.7 following a 24 h treatment with freshly prepared ZnO nanoparticles (ZnO I, 0 h), with ZnO suspension that were pre-incubated for 24 h at 37°C (ZnO I, 24 h), resuspended pellets of the 24 h pre-incubated suspension (P-ZnO I, 24 h) and the particle free supernatants collected after centrifugation of the pre-incubated suspension at 16.000 g (S-ZnO I, 24 h). All respective suspensions were prepared at 0.7 g/L, equaling the final treatment dose of 80 µg/cm². Staurosporine (STS, 24 h, 0.1 µM) was used as positive control. Data are expressed as percentage of total cell events (n = 2). Detection of Zn using a fluorescent indicator in RAW 264.7 cells treated with control medium (B), treated for 0 h with ZnO particles 80 µg/cm², freshly suspended (C), treated for 4 h with 5 µg/cm² (D) or 80 µg/cm² (E) freshly suspended ZnO particles or treated for 4 h with supernatant (F) as well as pellet (G) of a 80 µg/cm² ZnO suspension after preincubation for 24 h at 37°C. Original magnification 100×.</p

Cytotoxic effects of a panel of ZnO particles in RAW 264.7 macrophages.

<p>(A) Cell viability determined by WST-1 assay after 4 h treatment with four different ZnO particles at the indicated concentrations. Data are expressed as a percentage of the non-treated control cells, and represent three independent experiments (i.e. n = 3). As positive control, cells were treated for 4 h with 1 µM staurosporine (STS). (B) FACS analysis after 7-AAD staining revealing cells with hypodiploid DNA content after 4 h treatment with the panel of ZnO particles or staurosporine. Data are expressed as percentage of total cell events (n = 3).</p

Representative Scanning Electron Microscopy images of the four ZnO samples used in present study.

<p>Samples were analyzed by a LEO (Zeiss) 1530 Scanning Electron Microscope at a magnification of 10,000× as well as 50,000× (inserts).</p

DLS curves of ZnO suspensions in complete cell culture medium.

<p>(A) Representative data of freshly prepared suspensions of the four ZnO samples. (B) Comparison of freshly suspended ZnO I versus complete suspension or resuspended pellet of ZnO I after 24 h incubation at 37°C. Data were obtained with a Beckmann Coulter Delsa Nano C.</p

Z.average, minimum and maximum values as well as polydispersity index (PDI) of different ZnO suspensions.

<p>The suspension used for these analyses, were prepared in an identical manner to those that were used in the cell treatments (i.e. in complete cell culture medium and sonicated). Data represent three repeated measurements of two independent samples, and were obtained using a Beckmann Coulter Delsa Nano C.</p

Activated caspase-3 and DNA staining in RAW 264.7 macrophages after 4 h treatment with ZnO or staurosporine (STS).

<p>Representative images are shown for activated caspase-3 (first column) and corresponding DNA staining with Hoechst 33342 (second column). To control for unspecific staining, CC-3 antibody was substituted by IgG antibody (third column) and shown with the corresponding Hoechst 33342 staining (fourth column). Original magnification 400×.</p

Bone marrow-derived macrophages of Nrf2^−/− versus wt animals were investigated following 4 h ZnO treatment.

<p>(A) Cell viability (WST-1 assay) (n = 3). (B) FACS analysis to detect the percentage of cells with hypodiploid DNA after 7-AAD staining.</p