Using the Microcyte Flow Cytometer To Monitor Cell Number, Viability, and Apoptosis in Mammalian Cell Culture

Harding, C.L.; Lloyd, D. R.; McFarlane, Caroline M.; Al‐Rubeai, Mohamed

doi:10.1021/bp0000813

Cited by 31 publications

(18 citation statements)

References 47 publications

(58 reference statements)

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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

193

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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Section: Introductionmentioning

confidence: 99%

Inertial microfluidics for sheath-less high-throughput flow cytometry

Bhagat

Kuntaegowdanahalli

Kaval

et al. 2009

Biomed Microdevices

193

157

View full text Add to dashboard Cite

show abstract

“…This rapid method has the advantage that viable and dead cells can at least be distinguished objectively from debris on the basis of size and granularity, thus overcoming one problem of inter-observer variation. Furthermore a large number of particles can easily be counted, thus reducing sampling error and producing data with higher statistical validity (Al-Rubeai et al 1997;Harding et al 2000;Mukwena et al 2003).…”

Section: Introductionmentioning

confidence: 99%

NucleoCounter—An efficient technique for the determination of cell number and viability in animal cell culture processes

et al. 2006

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The NucleoCounter is a novel, portable cell counting device based on the principle of fluorescence microscopy. The present work establishes its use with animal cells and checks its reliability, consistency and accuracy in comparison with other cytometric techniques. The main advantages of this technique are its ability to handle a large number of samples with a high degree of precision and its simplicity and specificity in detecting viable cells quantitatively in a heterogeneous culture. The work addresses and overcomes the problems of subjectivity, and some of the inherent sampling errors associated with using the traditional haemocytometer and Trypan Blue exclusion method. NucleoCounter offers reduced intra-and inter-observer variation as well as consistency in repetitive analysis that establishes it as an efficient and highly potential device for at-line monitoring of animal cell processes. Furthermore, since the only manual steps required are sample aspiration and mixing with two reagents, it is feasible that the whole method could be automated and brought on-line for process monitoring and control.

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“…The isolated cells in a linear stream very quickly (up to 10 4 -10 5 cells/s) cross an area irradiated with one or more elliptically-focused laser beams, and fluorescent and/or forward and sideways scattering light is recorded with several photodetectors with spectral filters. This technique is widely used in basic and clinical research to provide, among other applications, rapid analysis of large populations of cells; information on cell sizes and shapes; detection of rare cells; and evaluation of drugcell interactions [Frazer, 2000;Harding et al, 2000]. Image analyzers (e.g., CAS 200, Becton Dickinson Immunocytometry Systems; and ImageStream, Amnis Corp.) have significantly extended the ability of FC to image cells in flow with a reasonable resolution of $1 mm and a flow velocity up to 5 m/s [Tibbe et al, 2002;Kubota, 2003;George et al, 2004].…”

mentioning

confidence: 99%

In vivo photothermal flow cytometry: Imaging and detection of individual cells in blood and lymph flow

Zharov

Galanzha

Tuchin

2006

J of Cellular Biochemistry

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Flow cytometry is a well-established, powerful technique for studying cells in artificial flow in vitro. This review covers a new potential application of this technique for studying normal and abnormal cells in their native condition in blood or lymph flow in vivo. Specifically, the capabilities of the label-free photothermal (PT) technique for detecting and imaging cells in the microvessel network of rat mesentery are analyzed from the point of view of overcoming the problems of flow cytometry in vivo. These problems include, among others, the influences of light scattering and absorption in vessel walls and surrounding tissues, instability of cell velocity, and cells numbers and positions in a vessel's cross-section. The potential applications of this new approach in cell biochemistry and medicine are discussed, including molecular imaging; studying the metabolism and pathogenesis of many diseases at a cellular level; and monitoring and quantifying metastatic and apoptotic cells, and/or their responses to therapeutic interventions (e.g., drug or radiation), in natural biological environments. J. Cell.

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Using the Microcyte Flow Cytometer To Monitor Cell Number, Viability, and Apoptosis in Mammalian Cell Culture

Cited by 31 publications

References 47 publications

Inertial microfluidics for sheath-less high-throughput flow cytometry

Inertial microfluidics for sheath-less high-throughput flow cytometry

NucleoCounter—An efficient technique for the determination of cell number and viability in animal cell culture processes

In vivo photothermal flow cytometry: Imaging and detection of individual cells in blood and lymph flow

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