Cell-cycle analysis of asynchronous populations.

Michael G Ormerod

doi:10.1385/1-59259-773-4:345

Home/Archive/PubMed

PubMed · 14976376

Cell-cycle analysis of asynchronous populations.

Michael G Ormerod

View original source ↗Export metadata ↓

Abstract

Cells are incubated continuously in bromodeoxyuridine (BrdUrd), which is incorporated into cells synthesizing DNA. At intervals, cells are harvested and nuclei are prepared and stained with a bis-benzimidazole, Hoechst 33258, and propidium iodide. In the flow cytometer, the dyes are excited by UV and blue and red fluorescences recorded. BrdUrd quenches the blue fluorescence of the Hoechst dye. The degree of quenching records the progress of the cell through S phase(s); the red (PI) fluorescence yields the cell cycle phases. By this means, the progress of cells around the cell cycle can be followed and the effects of cytotoxic drugs, radiation, and other treatments observed.

Explore related subjects

Bisbenzimidazole Bromodeoxyuridine Cell Cycle Cell Division Cell Nucleus Coloring Agents Detergents Fibroblasts Flow Cytometry Humans Propidium Time Factors Journal Article Research Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Michael G Ormerod. 2004. Cell-cycle analysis of asynchronous populations.. https://doi.org/10.1385/1-59259-773-4%3A345

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Studies on the interactions between DNA and PAMAM with fluorescent probe [Ru(phen)2 dppz]2+.

The interactions between plasmid DNA and cationic polymers are of interest for their potential biological applications. In this paper, the interactions of DNA with polyamidoamine (PAMAM) dendrimer were studied by fluorescence spectroscopy and stopped-flow technique. A rapid and reproducible fluorescent assay method had been developed for assessing PAMAM and DNA interactions using [Ru(phen)2 dppz]2+ as a probe. We further studied the kinetics of PAMAM binding to DNA and the reverse process of DNA dissociation from the complexes. The results indicated that DNA condensation was the rate-determining step during the complexation process, while DNA unfolding and expansion was the rate-determining step during the DNA dissociation process. At N/P ratios before reaching the thermodynamically most stable state, the complexes of DNA and PAMAM were incompact and could dissociate to some extent. And at N/P ratios above 2.0, DNA was fully condensed by PAMAM and dissociation was increasingly difficult. These results provided some useful instructions for self-assembling and disassembling of DNA as well as efficient gene delivery applications.

Bisbenzimidazole↗

PubMed2006

Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons.

Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.

Bisbenzimidazole↗

PubMed2005

Assay for the quantification of small-sized fragmented genomic DNA.

This paper presents a simple assay for the direct quantification of small-sized (up to 1000 bp) fragmented DNA based on the differential separation between large- and small-sized DNA by polyethylene glycol precipitation. The assay can be applied to as low as 1 microg ml(-1) DNA and has a very low DNA detection limit (0.01 microg ml(-1) fragmented DNA), which can be further lowered at least 10-fold with anion exchange chromatography. The assay quantifies for the first time a more direct indicator of the extent of DNA damage than the usually assessed DNA nicks and base modifications, since small-sized fragmented DNA represents an unrepairable DNA damage of necrotic and apoptotic events that are related to normal and abnormal biological conditions.

Bisbenzimidazole↗