Automated real-time measurements of leukocyte chemotaxis.

We have previously described an automated system (ECIS/taxis) for measuring chemotactic movement of Dictyostelium amoebae in a folic acid gradient [Hadjout, N., Laevsky, G., Knecht, D.A. and Lynes, M.A., 2001. Automated real-time measurement of chemotactic cell motility. Biotechniques 31, 1130-1138.]. In the ECIS/taxis system, cells migrate in an under-agarose environment, and their position is monitored by determining the impedance change caused by cells crawling onto the surface of an electrode. In this report, we show that chemotaxis of primary and immortalized leukocytes in response to complement (C5a) could be measured using the ECIS/taxis system. Several modifications to the design of the target electrode were tested, and a linear electrode perpendicular to the direction of movement was found to increase the sensitivity and reliability of the assay. Using the optimized ECIS/taxis assay, the dose response of neutrophils and WBC 265-9C cells was established and compared to the Boyden chamber assay. The ECIS/taxis assay system can be used to compare the movement of different cell types, to assess the effect of complex chemotactic gradients, or to determine the effects of pharmaceuticals on chemotactic motility.

Shrinking the biologic world--nanobiotechnologies for toxicology.

Although toxicologic effects need to be considered at the organismal level, the adverse events originate from interactions and alterations at the molecular level. Cellular structures and functions can be disrupted by modifications of the nanometer structure of critical molecules; therefore, devices used to assess biologic and toxicologic processes at the nanoscale will allow important new research pursuits. In order to properly assess alterations at these dimensions, nanofabricated tools are needed to detect, separate, analyze, and manipulate cells or biologic molecules of interest. The emergence of laser tweezers, surface plasmon resonance (SPR), laser capture microdissection (LCM), atomic force microscopy (AFM), and multi-photon microscopes have allowed for these assessments. Micro- and nanobiotechnologies will further advance biologic, clinical, and toxicologic endeavors with the aid of miniaturized, more sensitive devices. Miniaturized table-top laboratory equipment incorporating additional innovative technologies can lead to new advances, including micro total analysis systems (microTAS) or "lab-on-a-chip" and "sentinel sensor" devices. This review will highlight several devices, which have been made possible by techniques originating in the microelectronics industry. These devices can be used for toxicologic assessment of cellular structures and functions, such as cellular adhesion, signal transduction, motility, deformability, metabolism, and secretion.

Mechanisms of anemia in SHP-1 protein tyrosine phosphatase-deficient "viable motheaten" mice.

OBJECTIVE: Viable motheaten mice (abbreviated gene symbol me(v)) are deficient in SHP-1, a critical negative regulator of signal transduction in hematopoietic cells. These mice exhibit severe immune dysfunction accompanied by hyperproliferation of myeloid cells, widespread inflammatory lesions, and regenerative anemia. The aim of this study was to investigate the mechanisms underlying anemia in me(v)/me(v) mice. MATERIALS AND METHODS: Multiple hematologic parameters, osmotic fragility, and erythropoietin levels were measured to characterize the anemia in me(v)/me(v) mice. B-cell-deficient me(v)/me(v) Igh-6(null) mice were generated to assess the role of anti-erythrocyte antibodies. Coombs assays and flow cytometry were carried out for detection of anti-erythrocyte antibodies. Oxidant production by macrophages, glutathione levels, and lipid peroxidation products in erythrocytes were measured, as was the impact of oxidant on the ultrastructure of me(v)/me(v) erythrocytes. Erythroid maturation and erythrocyte plasma membrane integrity were assessed with flow cytometry by evaluating CD71 expression and annexin V labeling. RESULTS: The regenerative anemia of me(v)/me(v) mice was associated with erythrocyte changes that were independent of the presence of anti-erythrocyte antibodies. Erythrocytes from me(v)/me(v) mice had increased fragility and heightened susceptibility to oxidant damage. Macrophages from me(v)/me(v) mice demonstrated a higher basal level of oxidant production and enhanced production after stimulation. Oxidant damage in me(v)/me(v) erythrocytes was evidenced by a significant elevation of lipid peroxidation and diminished levels of glutathione. CONCLUSION: Our results support the hypothesis that as a consequence of severe inflammatory disease, me(v)/me(v) erythrocytes are subject to exceptionally high oxidative stress resulting in oxidation of phospholipids in the erythrocyte membrane with subsequent hemolysis.