Multiparametric Cell Cycle Analysis by Automated Microscopy

Abstract

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan TM reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells. (Journal of Biomolecular

In vitro structure-toxicity relationship of chalcones in human hepatic stellate cells

“…Nuclear DNA-staining allowed us to determine the size/area of the nucleus, as well as the DNA-content and thereby the cell cycle state of individual cells (Gasparri et al, 2006). In eukaryotic cells, actin fulfills very diverse and crucial functions (Field and Lénárt, 2011).…”

“…distribution. An increase of the ratio indicates an arrest in G1/G0 whereas a G2/M arrest is reflected by a decrease of this ratio (Gasparri et al, 2006).…”

Assessment of Cell Cycle Inhibitors by Flow Cytometry

“…Enzymatic treatment with DNase I / exonuclease III against A-T hypersensitive enzyme sites by digestion at 37 ° C of generate single stranded DNA to exposure incorporated BrdU to monoclonal antibody. This method has proved to be of particular interest for its ability to maintain antigenicity and morphology, allowing to follow additional cellular parameter such as cell signalling events (Gasparri et al, 2006). Additional methods were reported in the literature using treatment with high-energy radiation for the generation of DNA breaks by photolysis followed by anti-BrdU antibody (Leif et al, 2006).…”

“…The denaturation of DNA can be varied with time, only partially allowing the use of DNA probes staining such as propidium iodide (PI) , 7-aminoactynomycin D, TOPRO-3, which interacts DNA requiring double helix conformation. The use of acid denaturation may not be the most appropriate method if it is necessary to maintain cellular (scatters) morphology, surface antigenicity or cell constituents such as cyclins (Faretta et al, 1998) or phosphorylation of signaling proteins (Gasparri et al, 2006) during multiparameter analysis. 3.…”

Comparison of Flow Cytometry and Image-Based Screening for Cell Cycle Analysis

“…Similar image based approaches were presented in [2][3]. Here we quantitatively compare FC and IBHCS using automatic gate selection.…”