Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system.

A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment.

A single-fiber in vitro motility assay. In vitro sliding velocity of F-actin vs. unloaded shortening velocity in skinned muscle fibers.

We describe an approach that allows us to form a micro in vitro motility assay with as little myosin as can be retrieved from a short (approximately 10 mm) segment of a single skinned skeletal muscle fiber (diameter some 100 microm). Myosin is directly extracted from the single fiber segment by a high ionic strength solution in the presence of MgATP, and the extracted myosin is immediately applied to a miniaturized flow cell that has been pretreated with BSA. The observed sliding velocities of fluorescently labeled F-actin are essentially identical with those reported in the literature. Since at the single fiber level most muscle fibers contain only a single myosin heavy chain isoform this approach allows us to determine without additional purification steps, the sliding velocity driven by myosins with different heavy chain isoforms. In addition, this approach can be used to directly correlate under identical experimental conditions unloaded shortening velocity measured in segments of skinned muscle fibers with the in vitro sliding velocity of fluorescently labeled F-actin by extraction of myosin from the same skinned fibers. Such direct correlation was performed with different myosin heavy chain isoforms as well as at different temperatures and ionic strengths. Under all conditions studied, unloaded shortening velocity was 4- to 8-fold faster than sliding velocity in the motility assay even at high temperature (22 degrees C) and ionic strengths >50 mM. This suggests that sliding velocity in the motility assay is limited by additional factors beyond those thought to limit velocity of unloaded shortening in muscle fibers. One such factor might be unspecific ionic interactions between F-actin and the substrate in the motility assay resulting in somewhat higher sensitivity for ionic strength of sliding velocity in the motility assay. This might become of special relevance when using in vitro sliding velocity in assessing functional consequences of mutations involving charged residues of actin or myosin.