title: Miniaturizing bromodeoxyuridine incorporation enables the usage of flow cytometry for cell cycle analysis of adherent tissue culture cells for high throughput screening.
abstract: Analysis of cell cycle progression by 5-bromo-2'-deoxyuridine (BrdU) incorporation is commonly used for evaluating the mode of action of anticancer drugs, but usually requires a high number of cells and large amounts of monoclonal antibodies. In addition, manual sample handling is not suitable for high throughput. To circumvent these limitations, we have developed a miniaturized method to measure BrdU incorporation into DNA directly in 96-wells plates. Adherent cells were grown in 96-well plates in the absence or presence of compounds of interest. After BrdU pulse labeling or pulse chase, cells were harvested, transferred to polymerase chain reaction (PCR) V-bottom plates, and fixed by adding methanol. DNA denaturation was performed directly in the plates by heat using a PCR thermocycler. BrdU incorporation was detected by indirect immunocytochemical staining, and cellular DNA was counterstained using propidium iodide. Samples were acquired by a BD FACSCalibur with BD Multiwells Auto sampler or BD HTS. We defined a dynamic range of the optimal cell number, for which cells maintained exponential growth up to 72 h. The assay was robust up to 30,000 cells per well. BrdU dot plots of cell cycle phases showed an excellent separation of cell populations, and DNA histograms showed a low coefficient of variation. Thermal denaturation was suitable for 96-well plates to detect BrdU incorporation with a good signal-to-noise ratio, and cluster analysis allowed fingerprint readouts for drug sensitivity and mechanism of action as exemplified for paclitaxel and doxorubicin. This method provided rapid high-throughput BrdU/DNA content analysis with high accuracy and reproducibility, accompanied by a reduction in reagent consumption. A critical step was identified as the standardization of DNA denaturation using a PCR thermocycler. Here,we show some applications of this method for cell cycle studies in drug discovery.
authors.affiliation: Cell Biology Department, Nerviano Medical Sciences Srl, Nerviano, Italy. paolo.cappella@nervianoms.com
authors.forename: Paolo
authors.initials: P
authors.lastname: Cappella
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Giorgini
authors.forename: Claudia
authors.initials: C
authors.lastname: Ernestina Re
authors.forename: Paolo
authors.initials: P
authors.lastname: Ubezio
authors.forename: Marina
authors.initials: M
authors.lastname: Ciomei
authors.forename: Jürgen
authors.initials: J
authors.lastname: Moll
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000032:analysis*; D002452:Cell Count; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D046509:Tissue Culture Techniques
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D001973:Bromodeoxyuridine