Flow Cytometry

Detection of hTERT protein by flow cytometry

by Ased Ali

Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated... more
Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and are unable to isolate specific cell sub-populations. This report describes the development and validation of a cellular, immunofluorescence-based flow cytometry assay that allows detection of intranuclear hTERT while maintaining identifiable cell population characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differentiation experiments and in primary cells, a good correlation existed between hTERT expression measured by flow cytometry and telomerase activity detected by the telomeric repeat amplification protocol (TRAP). The method developed offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investigations, facilitating the analysis of hTERT expression in mixed cell populations.

Influence of geometry on hydrodynamic focusing and long-range fluid behavior in PDMS microfluidic chips

by James Hoyland

Details of hydrodynamic focusing in a 2D microfluidic channel-junction are investigated experimentally and... more Details of hydrodynamic focusing in a 2D microfluidic channel-junction are investigated experimentally and theoretically, especially the effect on the focusing width of volumetric flow ratio r between main and side channels, as well as angle θ between channels. A non-linear relationship is observed where the focus width decreases rapidly with increasing r and levels off at higher values. For the dependence on θ, results from both experiments and modeling show that an increased focusing effect is obtained as θ approaches 90°. Long-range focusing is explored along a 1 cm long channel and it is observed that in the middle section of the channel, a smaller θ induces less divergence. This effect is of importance for microfluidic systems utilizing hydrodynamic focusing in long, straight channels.

Flow cytometric analysis of benthic prokaryotes attached to sediment particles

by Stefano Amalfitano

Appropriate detachment treatments are required to analyze prokaryotes associated with streambed sediments by flow... more Appropriate detachment treatments are required to analyze prokaryotes associated with streambed sediments by flow cytometry. Using our previously optimized protocol, two groups of cells exhibiting different nucleic acid contents were easily detectable. However, the Nucleic Acid Double Staining assay proved that detachment procedures negatively affect the cell membrane integrity.

Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification

by Stefano Amalfitano

Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial... more Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an insolution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

Simulating cancer-cell kinetics after drug treatment: application to cisplatin on ovarian carcinoma

by Paolo Cappella

Istituto di Ricerche Farmacologiche “Mario Negri,” 20157 Milano, Italy

The kinetics of a cancer-cell population under the effects of an antitumoral drug is a topic of particular interest.... more The kinetics of a cancer-cell population under the effects of an antitumoral drug is a topic of particular interest. Its theoretical understanding, along with the improvement of experimental investigation techniques, can indeed play an important role in antitumoral therapies development. Starting from the analysis of flow-cytometric data, with the aid of computer simulation we are able to give a detailed, quantitative description of the main kinetic parameters describing drug action on cancer cells. In this paper we describe the main features of our investigation method, showing an application to Igrov-1 ovarian carcinoma cells treated with cisplatin. Intermitotic time of phases, cell-cycle delay, and block effects with consequent repair or cell mortality, in a wide range of drug doses and recovery times, are discussed and interesting information about cisplatin action is found.
© 1998 The American Physical Society

Genetic Characterization of New Brachypodium distachyon Populations from Diverse Geographic Regions in Turkey

by Siğnem ONEY

M. Tuna, İ. Nizam, S. Öney, G. Savaş and J. Vogel, 2011. 1st European Brachypodium Workshop, 19-21 October, Versailles-France

The small annual grass Brachypodium distachyon is rapidly emerging as a powerful model system for temperate grasses... more The small annual grass Brachypodium distachyon is rapidly emerging as a powerful model system for temperate grasses due to its biological, physical and genomic characteristics. The natural range of B. distachyon largely overlaps the ancestral range of cultivated small grains and Turkey lies in the center of that geographical area thus granting a great amount of genetic variability. The objective of this study was to establish and genetically characterize a large B. distachyon germplasm collection originating from diverse geographical regions in Turkey. Seeds from approximately 20 individual plants were collected from each of the 118 locations throughout Turkey. Flow cytometry was used to determine the ploidy of representatives from each location. Seventy five of the populations contained only diploid plants, 35 of the populations contained only polyploid plants and 8 populations contained a mix of diploid and polyploid plants. To examine the genetic diversity of this new collection, a representative subset of 185 diploid individuals was genotyped using SSR markers. Our preliminary results show considerable genetic diversity both between and within populations. We also compared the SSR profiles of the new lines to previously examined lines. Significantly, some of the new accessions were contained in new clades indicating that we have not sampled Turkish B. distachyon diversity to saturation. Future plans for this collection include phenotypic characterization with an emphasis on biotic and abiotic stress. We also plan to develop recombinant inbred lines from divergent lines.

The Active Human Gut Microbiota Differs from the Total Microbiota

by Giuseppe D'Auria

Francesc Peris-Bondia, Amparo Latorre, Alejandro Artacho, Andrés Moya, Giuseppe D'Auria

PLoS ONE 6(7): e22448. doi:10.1371/journal.pone.0022448

The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between... more The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis.

Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration.

Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

by João Loureiro

Sympatric diploid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the Eastern Alps are separated along an altitudinal gradient

by Christian Lettner

Schönswetter P., Lachmayer M., Lettner C., Prehsler D., Rechnitzer S., Reich D.S., Sonnleitner M., Wagner I., Hülber K., Schneeweiss G.M., Trávníček P. & Suda J. (2007): Sympatric diploid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the Eastern Alps are separated along an altitudinal gradient. Journal of Plant Research 120: 721-725.

[Sorry! PDF not available for download. (© The Botanical Society of Japan and Springer 2007)]

We explored the fine-scale distribution of cytotypes of the mountain plant Senecio carniolicus along an altitudinal... more We explored the fine-scale distribution of cytotypes of the mountain plant Senecio carniolicus along an altitudinal transect in the Eastern Alps. Cytotypes showed a statistically significant altitudinal segregation with diploids exclusively found in the upper part of the transect, whereas diploids and hexaploids co-occurred in the lower range. Analysis of accompanying plant assemblages revealed significant differences between cytotypes along the entire transect but not within the lower part only, where both cytotypes co-occur. This suggests the presence of ecological differentiation between cytotypes with the diploid possessing the broader ecological niche. No tetraploids were detected, indicating the presence of strong crossing barriers.

Ploidy levels among species of the “Oxalis tuberosa alliance” as inferred by flow cytometry.

by Eve Emshwiller

Emshwiller, Eve.  2002.  Annals of Botany. 89(6): 741-753.

The ‘Oxalis tuberosa alliance’ is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa ... more The ‘Oxalis tuberosa alliance’ is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa  Molina (Oxalidaceae), commonly known as ‘oca’. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C‐values in the alliance by estimating nuclear DNA contents of these accessions using chicken erythrocytes as internal standard. Ten Bolivian accessions of cultivated O. tuberosa were confirmed to be octoploid, with a mean nuclear DNA content of approx. 3·6 pg/2C. Two Peruvian wild Oxalis species, O. phaeotricha and O. picchensis, were inferred to be tetraploid (both with approx. 1·67 pg/2C), the latter being one of the putative progenitors of O. tuberosa  identified by chloroplast‐expressed glutamine synthetase data in prior work. The remaining accessions (from 78 populations provisionally identified as 35 species) were DNA diploid, with nuclear DNA contents varying from 0·79 to 1·34 pg/2C.