Separation of live cells in different phases of the cell cycle for gene expression analysis

Juan, Gloria; Hernando, Eva; Cordon‐Cardo, Carlos

doi:10.1002/cyto.10173

Cited by 30 publications

(24 citation statements)

References 17 publications

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“…The recovery ratio is defined as Recovery Ratio (%) ϭ ͑N r /N f ͒ ϫ 100 (2) where N r is the number of cells in the permeate solution after the permeation of HSA solution. The filtration experiments were performed at 25 Ϯ 0.5°C.…”

Section: Methodsmentioning

confidence: 99%

“…Cell separation is typically accomplished by centrifugation, 1 fluorescence-activated cell sorting (FACS), 2 magnetic cell selection, [3][4][5][6] affinity column chromatography, or membrane filtration. The centrifugal separation of cells is a typical method used to separate platelets, leukocytes, red blood cells, and non-blood cells.…”

Section: Introductionmentioning

confidence: 99%

“…The most highly purified cellular preparations are obtained by using FACS in conjunction with a fluorescently labeled antibody for the cell surface marker. Juan et al 2 investigated cell separation using FACS in different phases of cell cycle by sorting G 1 and G 2 /M cells, followed by RNA isolation from the sorted cells, because homogeneity of cell population is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. They found that sorted living G 1 and G 2 /M cells, analyzed by immunocytochemistry and laser-scanning cytometry, showed strong enrichment.…”

Section: Introductionmentioning

confidence: 99%

“…They found that sorted living G 1 and G 2 /M cells, analyzed by immunocytochemistry and laser-scanning cytometry, showed strong enrichment. 2 However, FACS cannot be applied to clinical applications due to difficulties with sterility and the excessive time needed to purify sufficient quantities of specific cells such as CD34 ϩ cells.…”

Section: Introductionmentioning

confidence: 99%

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Cell separation of hepatocytes and fibroblasts through surface‐modified polyurethane membranes

Higuchi

Tsukamoto

2004

J Biomedical Materials Res

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The separation of fibroblast cells (L929 cells) and hepatocytes was investigated by using unmodified and surface-modified polyurethane (PU) foaming membranes (pore size of 12 microm) by the incorporation of various functional groups. L929 cells permeated more readily than hepatocytes, and very few populations of hepatocytes (<5%) permeated through the membranes. This result was thought to be due to the smaller cell size of the L929 cells (5-10 microm) relative to the hepatocytes (15-30 microm). The larger hepatocytes were thought to plug the pores of the membranes. A good cell separation between L929 cells and hepatocytes was achieved when the cell mixture permeated through the negatively charged PU membranes. The negatively charged membranes were thought to enhance the permeation of L929 cells because of the electrostatic repulsion between the membranes and negatively charged cells. On the other hand, the hepatocytes were unable to permeate through the membranes because of the sieve effect caused by their large cell size. The separation of hepatocytes isolated from mice at different ages was also accomplished by permeating the cell mixture through unmodified and surface-modified PU membranes.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cell separation of hepatocytes and fibroblasts through surface‐modified polyurethane membranes

Higuchi

Tsukamoto

2004

J Biomedical Materials Res

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“…2 The differentiation between tumor cells actively proliferating and those committed to apoptosis is important to the study of cancer. The use of stains such as the combination of Hoescht 33342, propidium iodide and fluorescent anti-cyclin antibody 3 can allow for a multiparametric cell death and cell cycle analysis. However, these protocols are limited by requiring the sample to be fixed, thereby preventing live cell analysis.…”

Section: Introductionmentioning

confidence: 99%

Properties of cells through life and death – an acoustic microscopy investigation

et al. 2015

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Current methods to evaluate the status of a cell are largely focused on fluorescent identification of molecular biomarkers. The invasive nature of these methods -requiring either fixation, chemical dyes, genetic alteration, or a combination of these -prevents subsequent analysis of samples. In light of this limitation, studies have considered the use of physical markers to differentiate cell stages. Acoustic microscopy is an ultrahigh frequency (>100 MHz) ultrasound technology that can be used to calculate the mechanical and physical properties of biological cells in realtime, thereby evaluating cell stage in live cells without invasive biomarker evaluation. Using acoustic microscopy, MCF-7 human breast adenocarcinoma cells within the G1, G2, and metaphase phases of the proliferative cell cycle, in addition to early and late programmed cell death, were examined. Physical properties calculated include the cell height, sound speed, acoustic impedance, cell density, adiabatic bulk modulus, and the ultrasonic attenuation. A total of 290 cells were measured, 58 from each cell phase, assessed using fluorescent and phase contrast microscopy. Cells actively progressing from G1 to metaphase were marked by a 28% decrease in attenuation, in contrast to the induction of apoptosis from G1, which was marked by a significant 81% increase in attenuation. Furthermore late apoptotic cells separated into 2 distinct groups based on ultrasound attenuation, suggesting that presently-unidentified sub-stages may exist within late apoptosis. A methodology has been implemented for the identification of cell stages without the use of chemical dyes, fixation, or genetic manipulation.

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Induction of DNA damage response by the supravital probes of nucleic acids

et al. 2009

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The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV), and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981), and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2, or p53 phosphorylation. However, pronounced ATM, Chk2, and p53 phosphorylation and perturbed G 2 progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2, and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2, and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G 2 M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5, and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis. ' 2009 International Society for Advancement of Cytometry

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Separation of live cells in different phases of the cell cycle for gene expression analysis

Cited by 30 publications

References 17 publications

Cell separation of hepatocytes and fibroblasts through surface‐modified polyurethane membranes

Cell separation of hepatocytes and fibroblasts through surface‐modified polyurethane membranes

Properties of cells through life and death – an acoustic microscopy investigation

Induction of DNA damage response by the supravital probes of nucleic acids

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