Multiplexed approaches correlating mitochondrial health to cell health using microcapillary cytometry.

Mitochondria are critical cellular organelles that play a fundamental role in cellular metabolism and oxidative stress and are well known to trigger multiple cell death pathways. The study of sequence of mitochondrial events as it relates to apoptotic/cell death events can provide critical insights into mechanism of cellular homeostasis, stress and death. Availability of rapid and simplified cytometric testing methods for evaluating mitochondrial changes, apoptosis and cell death in parallel can greatly enhance our understanding of mechanism of compound action. In this study, we investigated a series of compounds to evaluate apoptotic/cell death effects in context of mitochondrial changes using plate-based assays on Guava® easyCyte systems. Studies utilized multiplexed assays for mitochondrial membrane potential changes and apoptosis/cell death markers and allowed for easy identification of hit compounds. Dose and time response studies with Niclosamide, an anti-helmintic drug and comparison of effects with Gambogic acid and celastrol demonstrated early and significant mitochondrial impacts for niclosamide and gambogic acid. No apoptotic or cell death impacts were observed in parallel at low doses/short times of incubation for niclosamide, while increased time with niclosamide caused increase in mitochondrial, apoptotic and cell death response. The method demonstrates great power in being able to distinguish between potency of compounds and conditions in modulating mitochondrial/apoptotic changes. The simplicity of the assays described coupled with the ease of use of plate based microcapillary cytometry can provide researchers valuable tools to obtain a more comprehensive insight into how compounds modulate mitochondria and its relationship with subsequent apoptosis/cell death pathways.

Simplified evaluation of apoptosis using the Muse cell analyzer.

The degree of apoptosis in a cell population is an important parameter of cell health and is characterized by distinct morphological changes. Current methods of accurate detection and measurement of cellular apoptosis require expensive and complicated instrument platforms and expertise. The Muse Cell Analyzer is a unique instrument that enables multidimensional cell health analysis on a single platform. In this study, we used the Muse Cell Analyzer for apoptosis studies using the Muse Annexin V & Dead Cell Assay. The assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells. The results obtained from Muse Cell Analyzer were compared with traditional methods for apoptosis analysis. Our results indicate that Muse Annexin V & Dead Cell Assay and software module enabled the acquisition of accurate and highly precise measurements of cellular apoptosis. The assay is versatile and works with both suspension and adherent cell lines and multiple treatment conditions.

Thiol-reactive dyes for fluorescence labeling of proteomic samples.

Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA and BODIPY Fl C(1)-IA were highly specific for cysteine residues and showed little or no nonspecific labeling even at very high dye:thiol ratios. These dyes also showed minimal effects on pI's of standard proteins. Some iodoacetamide dyes, (5-TMRIA and eosin-5-iodoacetamide) and some maleimide dyes (ThioGlo I and Rhodamine Red C(2) maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2-carboxyethyl)phosphine (TCEP); interactions between TCEP and dye were also observed. Thiourea, an important component of sample solubilization cocktails, inhibited labeling of proteins with iodoacetamide dyes but not with maleimide dyes. Maleimide dyes may serve as an alternative for labeling proteins where it is essential to have thiourea in the solubilization buffer. Covalent derivatization by BODIPY TMR cadaverine IA, BODIPY Fl C(1)-IA or Rhodamine Red C(2) maleimide was also demonstrated to be compatible with in-gel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionization-mass spectrometry and allowed successful protein identification.