Current status of flow cytometry in cell and molecular biology

Boeck, Guenther

doi:10.1016/s0074-7696(01)04006-2

Cited by 33 publications

(21 citation statements)

References 336 publications

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“…Flow cytometry allows characterization of nuclear DNA content by measuring the degree of fluorescence reflection of stained DNA in single cells. FCM is a well-proven method that is commonly used in life sciences for diagnosis or cell function analyses (Boek 2001). In addition, it has been widely applied for largescale comparative analyses of genome-size evolution, taxonomic identification and delineation, and studies of polyploids, reproductive biology, and experimental evolution in both plants and insects (Kron et al 2007).…”

Section: Ploidy Level Analysismentioning

confidence: 99%

Diploid males, diploid sperm production, and triploid females in the ant Tapinoma erraticum

Cournault

Aron

2009

Naturwissenschaften

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Under complementary sex determination (CSD), females of Hymenoptera arise from diploid, fertilized eggs and males from haploid, unfertilized eggs. Incidentally, fertilized eggs that inherit two identical alleles at the CSD locus will develop into diploid males. Diploid males are usually unviable or sterile. In a few species, however, they produce diploid sperm and father a triploid female progeny. Diploid males have been reported in a number of social Hymenoptera, but the occurrence of triploid females has hardly ever been documented. Here, we report the presence of triploid females, diploid males, and diploid sperm (produced by diploid males and stored in queen spermathecae) in the ant Tapinoma erraticum. Moreover, we show variations in the frequency of triploids among female castes: Triploid females are more frequent among workers than virgin queens; they are absent among mated, reproductive queens. The frequency of triploid workers also varies between populations and between nests within populations.

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Section: Ploidy Level Analysismentioning

confidence: 99%

Diploid males, diploid sperm production, and triploid females in the ant Tapinoma erraticum

Cournault

Aron

2009

Naturwissenschaften

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“…By capturing the scattered light and fluorescence emission, physical and chemical properties of the cells can be determined and cells can be sorted and counted. This high throughput technique is used in a variety of biomedical (Harding et al 2000;Boeck 2001) and clinical diagnostic applications (Stein et al 1992;Fenili and Pirovano 1998) including bacterial analysis (Gunasekera et al 2000), analysis of cell cycle and apoptosis (Darzynkiewicz et al 2001), detection of minimal residual disease (MRD) in leukemia cases (Deptala and Mayer 2001), and enumeration of CD4+ T-cells in HIV diagnosis (Cheng et al 2007). Additionally, flow cytometers can be used purely in cell sorting applications (Melamed et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Inertial microfluidics for sheath-less high-throughput flow cytometry

Bhagat

Kuntaegowdanahalli

Kaval

et al. 2009

Biomed Microdevices

196

157

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Flow cytometer is a powerful single cell analysis tool that allows multi-parametric study of suspended cells. Most commercial flow cytometers available today are bulky, expensive instruments requiring high maintenance costs and specially trained personnel for operation. Hence, there is a need to develop a low cost, portable alternative that will aid in making this powerful research tool more accessible. In this paper we describe a sheath-less, on-chip flow cytometry system based on the principle of Dean coupled inertial microfluidics. The design takes advantage of the Dean drag and inertial lift forces acting on particles flowing through a spiral microchannel to focus them in 3-D at a single position across the microchannel cross-section. Unlike the previously reported micro-flow cytometers, the developed system relies entirely on the microchannel geometry for particle focusing, eliminating the need for complex microchannel designs and additional microfluidic plumbing associated with sheath-based techniques. In this work, a 10-loop spiral microchannel 100 microm wide and 50 microm high was used to focus 6 microm particles in 3-D. The focused particle stream was detected with a laser induced fluorescence (LIF) setup. The microfluidic system was shown to have a high throughput of 2,100 particles/sec. Finally, the viability of the developed technique for cell counting was demonstrated using SH-SY5Y neuroblastoma cells. The passive focusing principle and the planar nature of the described design will permit easy integration with existing lab-on-a-chip (LOC) systems.

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“…Hence, the isolation of yeast that express these proteins requires a nontraditional screening approach. To provide an alternative strategy, we investigated the utility of transient protein expression, protein expression analysis by flow cytometry, [25][26][27] and isolation of individual yeast cells by FACS. For these experiments, we used yEGFP (yeast enhanced green fluorescent protein) as reporter of gene expression.…”

Section: Resultsmentioning

confidence: 99%

“…This flow cytometry-based approach was chosen because of its potential to rapidly quantify the cellular fluorescence of individual yeast cells. [25][26][27] Moreover, the ability to isolate single fluorescent cells from a heterogeneous culture of yeast could potentially streamline the identification of hits. The method that we employed, outlined in Figure 3, comprised the following steps: 1) transformation of the cDNA library into yeast that harbor the tribrid-system plasmids; 2) incubation in liquid selection media for two days to expand the population of transformed cells; 3) a brief 4 h induction of protein expression to circumvent the toxicity associated with PTK expression, but also to activate the fluorescent reporter gene in cells that express these enzymes; 4) isolation of putative hits by FACS onto solid yeast media; and 5) further analysis of hits by replica plating onto solid counter-selection media that contained 5-FOA.…”

Section: Analysis Of Yeast Tribrid Systems By Flow Cytometry and Facsmentioning

confidence: 99%

Fluorescence‐Based Cloning of a Protein Tyrosine Kinase with a Yeast Tribrid System

Clark

Peterson

2005

ChemBioChem

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Post-translational modifications of proteins control myriad biological functions. However, relatively few methods exist for the identification of the enzymes that catalyze these modifications. To expand this repertoire, we report a yeast genetic approach that enables the identification of protein tyrosine kinases (PTKs) from cDNA libraries. Yeasts were transformed with four vectors encoding: 1) a potentially universal PTK substrate fused to the LexA DNA binding domain, 2) the Grb2-SH2 domain fused to the B42 activation domain, 3) a fluorescent reporter gene controlled by LexA DNA sites, and 4) a Jurkat cDNA library. Transient expression of PTKs, such as the lymphocyte-specific kinase Fyn, resulted in phosphorylation of the DNA-bound substrate, recruitment of the Grb2-SH2 domain, and activation of the fluorescent reporter gene. This brief induction of protein expression circumvented the potential toxicity of PTKs to the yeast. Fluorescence activated cell sorting (FACS) enabled isolation of PTKs, and these enzymes were further characterized by flow cytometry and immunoblotting. This approach provides a potentially general method for the identification and evaluation of enzymes involved in the post-translational modification of proteins.

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Current status of flow cytometry in cell and molecular biology

Cited by 33 publications

References 336 publications

Diploid males, diploid sperm production, and triploid females in the ant Tapinoma erraticum

Diploid males, diploid sperm production, and triploid females in the ant Tapinoma erraticum

Inertial microfluidics for sheath-less high-throughput flow cytometry

Fluorescence‐Based Cloning of a Protein Tyrosine Kinase with a Yeast Tribrid System

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