Volume Electron Research Articles

Clustering of spermatozoa examined through flow cytometry provides more information than the conventional assessment: a resilience to osmotic stress example.

Context Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods Spermatozoa were exposed to either isotonic (300mOsm/kg) or hypotonic (180mOsm/kg) media for 5 and 20min, and were then stained with SYBR14 and propidium iodide (PI). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+ /PI- ) or with different degrees of plasma membrane alteration (SYBR14+ /PI+ and SYBR14- /PI+ ). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P P P Conclusions Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation.

Leukotoxin fromAggregatibacter actinomycetemcomitanscauses shrinkage and P2X receptor-dependent lysis of human erythrocytes

Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat-in-toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α-haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA-mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA-mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS-exposure is preceded by increments in the intracellular Ca(2+) concentration ([Ca(2+)](i)). Interestingly, LtxA-mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA-induced [Ca(2+)](i) increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2-mediated auto- and paracrine signalling may prevent LtxA-mediated cell damage.

Abstract 4054: Cell viability, apoptosis and ploidy analyses on a multi-laser, versatile flow cytometry analyzer – the EclipseTM

Abstract Cell viability, apoptosis and ploidy analyses are essential tools in cancer research involving in vitro and in vivo studies. Assays for cell count and viability, apoptosis and cell cycle analysis are described in this report. Jurkat or Chinese hamster ovary cells were treated with various reagents such as anti-CD95, taxol, or brefaldin A to induce apoptosis. Apoptotic cells were labeled with Avenxin-V conjugated with Pacific Blue or FITC to determine externalization of phosphatidylserine to the cell surface. In combination with cell impermeant dyes, such as 7-aminoactinomycin D (7-AAD) or propidium iodide (PI), live, early apoptotic and late apoptotic/dead cells were labeled and analyzed on an Eclipse equipped with the 405 and 488 lasers. The iCyt® Eclipse TM is a compact bench top flow cytometry analyzer with an autoloader that supports a wide variety of multi-well plate and test tube formats. The system can be equipped with up to 4 lasers (405, 488, 561 and 642nm) and provides simultaneous detection of 5 colors, forward scatter, side scatter, electronic volume (EV) and cell concentration. The EV parameter enables accurate cell volume (size) measurement and allows normalization of fluorescent intensity on a per volume or surface area basis. Apoptotic cells were also detected with a phycoerythrin-labeled anti-active caspase 3 antibody after fixation and permeabilization. Cell cycle analysis with G0/G1, S and G2/M phase statistics plus cell size was achieved by labeling cells with 4′,6-diamidino-2-phenylindole (DAPI) in a cell permeabilization solution and analyzing on the Eclipse. Together with its intuitive software, the Eclipse flow cytometry analyzer provides a rapid, accurate and cost effective tool for most flow cytometry applications such as cell viability, apoptosis and cell cycle analysis. Note: For research use only. Not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2011-4054

Simultaneous Determination of Phenotypic Expression and Electronic Volume in Mouse Cells by the Cell Lab Quanta® SC-MPL Analyzer. (B210)

Abstract The Cell Lab Quanta® SC analyzer, a unique platform with two excitation light sources; a 488 nm diode laser and a HBO arc lamp, can simultaneously measure Electronic Volume (EV), side scatter (SS) and three color fluorescence. This system has an innovative multiplate loader (MPL), which can undertake automatic sample preparation and high throughput analysis. We have used this instrument to analyze cells from various murine tissues with the objective to describe and identify changes in cellular volume and morphological complexity during development, activation and differentiation. Immunophenotypic analysis of cells isolated from bone marrow, peripheral blood, spleen, thymus and lymph nodes of C57 BL/6 mice was done with conventional combinations of MABs conjugated with FITC, PE and PE cy-5. The MPL was used for sample preparation in 96 well plates. EV, which is not used at present as a relevant parameter in flow cytometry, is a more precise measure of cell size than the conventional Forward Scatter (FS). EV can be calibrated with NIST traceable beads or appropriate biological calibrators with known Mean Corpuscular Volume. We conclude that this new automated platform expands the possibilities for researchers by providing means for identifying and differentiating between different cell types on the basis of EV/SS and also by improving the throughput with rapid sample analysis.

Flow cytometric characterization of alveolar macrophages.

Phenotypes of lung free cells (FC) harvested from Fischer-344 rats by episodic lavage were characterized by flow cytometry. Parameters evaluated included electronic volume (EV), axial light loss (ALL), 90 degrees light scatter (LS), blue autofluorescence (BA), and green-yellow autofluorescence (G-YA). Three phenotypic populations, FC-A, FC-B, and FC-C were identified by their differing LS characteristics. FC-C represented 90% of the cells and were exclusively alveolar macrophages. Two subpopulations in FC-C, FC-CI and FC-CII, were further distinguished by their unique ALL features. The morphologic appearances of these subpopulations by light microscopy clearly differed in sorted preparations. Based on their patterns of autofluorescence, these FC-CI and FC-CII phenotypes were found to be composed of eight subpopulations. In FC populations harvested during further lavage episodes of the same lungs, the relative contributions of FC-CI to the FC-C subpopulation decreased as FC-CII correspondingly increased. This study demonstrates 1) that subpopulations of lavaged AM can be categorized according to their optical phenotypes by flow cytometry and 2) that the relative frequency of retrieval of some phenotypes depends on how exhaustively the lungs are lavaged. With regard to the latter, bronchoalveolar lavage does not randomly sample the underlying AM population in the alveolar compartment.