Search PubMed⌕ Search

PubMed · 14570213

Microfluidic device for single-cell analysis.

Abstract

We have developed a novel microfluidic device constructed from poly(dimethylsiloxane) using multilayer soft lithography technology for the analysis of single cells. The microfluidic network enables the passive and gentle separation of a single cell from the bulk cell suspension, and integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell. Various applications are demonstrated, including cell viability assays, ionophore-mediated intracellular Ca2+ flux measurements, and multistep receptor-mediated Ca2+ measurements. These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Aaron R Wheeler, William R Throndset, Rebecca J Whelan, Andrew M Leach, Richard N Zare, Yish Hann Liao, Kevin Farrell, Ian D Manger, Antoine Daridon. 2003-07-15. Microfluidic device for single-cell analysis.. https://doi.org/10.1021/ac0340758

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

The combined effects of two anti-aggregatory agents, alpha-cyclodextrin and Ca2+, on the refolding process of denatured alpha-amylase.

It is now a well-known fact that alpha-CD (alpha-cyclodextrin) and Ca(2+) are two important folding agents in the re-activation and refolding processes of many denatured enzyme solutions. Bacillus sp. alpha-amylase was extensively denatured in a 6 M guanidinium chloride solution overnight. Under suitable renaturation conditions, 20-30% more activity was recovered in the presence of various concentrations of alpha-CD (0-100 mM). Similarly, 25-30% more activity was recovered under the influence of different concentrations of Ca(2+) (0-100 mM). Regardless of these results, both alpha-CD and Ca(2+) at 100 mM concentrations were separately capable of preventing the extent of aggregate formation by 86 and 77% respectively. These observations clearly indicate that the effects of alpha-CD and/or Ca(2+) on the process of aggregation and refolding (activity recovery) are not synchronized: both agents mostly act as anti-aggregatory agents instead of acting as refolding agents.

Calcium↗

Alterations of FSH-stimulated progesterone production and calcium homeostasis in primarily cultured human luteinizing-granulosa cells induced by fenvalerate.

Fenvalerate, a synthetic pyrethroid, is widely used in agriculture and other domestic applications in China. Recently, Fenvalerate has been suspected to be one of the endocrine-disrupting chemicals (EDC). In this study, we investigated the effects of fenvalerate on follicle-stimulating hormone (FSH)-stimulated progesterone (P4) production by human ovarian luteinizing-granulosa cells (hGLCs). After 24 h incubation, fenvalerate inhibited FSH-stimulated P4 production. At the same time, FSH-stimulated cAMP also decreased. Due to calcium and Ca2+ -calmodulin (CaM) system involving gonadotropin-stimulated steroidogenesis by granulosa cells, we then evaluated the effects of fenvalerate on trifluoperazine (TFP)- and verapamil-driven FSH-stimulated P4 production. The results showed that calcium or calmodulin might play a role in fenvalerate-induced alterations in FSH-stimulated P4 biosynthesis. Then, the effects of fenvalerate on calcium homeostasis in hGLCs were studied. The result showed that 5 microM fenvalerate induced a slow increase in [Ca2+]i in hGLCs by using a fluorescent Ca2+ indicator fluo-3/AM. The changes in total concentration of CaM in hGLCs induced by fenvalerate were evaluated by a method of immunofluorescence. There is a significant increase in all treated groups. In summary, fenvalerate could inhibit FSH-stimulated P4 production. Also, fenvalerate interferes with calcium homeostasis in hGLCs. The effects of fenvalerate on FSH-stimulated ovarian steroidogenesis may be mediated partly through calcium signal.

Calcium↗

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist.

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Capsazepine may be mildly cytotoxic.

Calcium↗