A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death.

The temporal context of cell death decisions remains generally hidden in ensemble measurements with endpoint readouts. Here, we describe a method to extract event times from fluorescence time traces of cell death-related markers in automated live-cell imaging on single-cell arrays (LISCA) using epithelial A549 lung and Huh7 liver cancer cells as a model system. In pairwise marker combinations, we assess the chronological sequence and delay times of the events lysosomal membrane permeabilization, mitochondrial outer membrane permeabilization and oxidative burst after exposure to 58 nm amino-functionalized polystyrene nanoparticles (PS-NH2 nanoparticles). From two-dimensional event-time scatter plots we infer a lysosomal signal pathway at a low dose of nanoparticles (25 µg mL-1) for both cell lines, while at a higher dose (100 µg mL-1) a mitochondrial pathway coexists in A549 cells, but not in Huh7. In general, event-time correlations provide detailed insights into heterogeneity and interdependencies in signal transmission pathways.

Single Cell Microarrays Fabricated by Microscale Plasma-Initiated Protein Patterning (µPIPP).

Micropatterned arrays considerably advanced single cell fluorescence time-lapse measurements by providing standardized boundary conditions for thousands of cells in parallel. In these assays, cells are forced to adhere to defined microstructured protein islands separated by passivated, nonadhesive areas. Here we provide a detailed protocol on how to reproducibly fabricate high quality single cell arrays by microscale plasma-initiated protein patterning (µPIPP). Advantages of µPIPP arrays are the ease of preparation and the unrestricted choice of substrates as well as proteins. We demonstrate how the arrays enable the efficient measurement of single cell time trajectories using automated data acquisition and data analysis by example of single cell gene expression after mRNA transfection and time courses of single cell apoptosis. We discuss the more general use of the protocol for assessment of single cell dynamics with the help of fluorescent reporters.

Impact of Experimental Variables on the Protein Binding of Tigecycline in Human Plasma as Determined by Ultrafiltration.

Tigecycline, a tetracycline derivative, shows atypical plasma protein binding behavior. The unbound fraction decreases with increasing concentration at therapeutic concentrations. Moreover, uncertainty exists about the magnitude of tigecyline's protein binding in man. Unbound fractions between 2.5% and 35% have been reported in plasma from healthy volunteers, and between 25% and 100% in patients, respectively. In the present study, the protein binding of tigecycline has been investigated by ultrafiltration using different experimental conditions. Whereas temperature had only a marginal influence, the unbound fraction at 0.3/3.0 mg/L was low at pH 8.2 (9.4%/1.9%) or in unbuffered pooled plasma (6.3%/1.2%), compared with plasma buffered with HEPES to pH 7.4 (65.9%/39.7%). In experiments with phosphate buffer and EDTA, the concentration dependency was markedly attenuated or abolished, which is compatible with a cooperative binding mechanism involving divalent cations such as calcium. The unbound fraction in clinical plasma samples from patients treated with tigecycline was determined to 66.3 ± 13.7% at concentrations <0.3 mg/L compared with 41.3 ± 16.0% at >1 to <5 mg/L. To summarize, tigecycline appears to be only moderately bound to plasma proteins as determined by ultrafiltration, when a physiological pH is maintained.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.