The Cell Cycle Analysis Assay is a vital tool for assessing the effects of various compounds and treatments on the cell cycle. This assay provides valuable insights by measuring changes in DNA content, synthesis, and the number of actively dividing cells. It plays a crucial role in understanding how different agents influence the intricate dynamics of the cell cycle, making it an essential component of our research services.
This comprehensive assay involves the following key steps:
- Incorporation of EdU: To detect newly synthesized DNA, we employ a fluorescently labeled nucleoside analog, 5-ethynyl-2′-deoxyuridine (EdU), providing real-time insights into DNA replication.
- Fluorescent Labeling: Cells are carefully labeled with DNA-binding fluorescent dyes, including widely used options like propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), or Hoechst.
- Identification of Actively Dividing Cells: Immunostaining targeting phosphohistone H3 further enhances our capabilities. It allows us to identify cells that are actively undergoing division.
- Fluorescence Microscopy: The labeled cells are subjected to fluorescence microscopy, enabling us to precisely analyze their DNA content and synthesize invaluable data about their cell cycle stage.
Cells navigate through distinct phases during the cell cycle, encompassing G1, S, G2, and M phases. Our approach involves measuring the relative distribution of cells across these phases, providing valuable insights into how specific treatments impact cell cycle progression.
For instance, when assessing the effects of a treatment, we examine the proportion of cells in each phase. A compound that inhibits cell proliferation may lead to an increased presence of cells in the G1 phase. In contrast, a compound that stimulates cell growth could result in a higher proportion of cells in the S phase. Our methodology empowers us to precisely evaluate and quantify these changes using high-throughput microscopy to screen large libraries of compounds or drugs, facilitating a deeper understanding of treatment outcomes.
Assay summary
| DNA staining with one of the intercalating dyes (DAPI, Hoechst, PI, 7-AAD) | DNA staining (Hoeachst) + nucleotide incorporation (EdU) + mitosis marker (pH3 antibody) | |
| Working principle | The method involves the use of fluorescent dyes that bind to DNA, allowing to determine the DNA content of individual cells. This analysis helps identify the different phases of the cell cycle, such as G1, S, G2/M phases. | The method involves incorporation of EdU to estimate the number of cells in the S-phase, followed by the use of fluorescent DNA-binding dye (Hoechst) to identify cells in the G1 and G2 phases based on their DNA content. Additionally, the technique includes staining with a phospho-Histone 3 (pH3) antibody to differentiate cells in the M phase of the cell cycle |
| What is detected | fluorescence intensity of nuclei | fluorescence intensity of nuclei in 3 different channels |
| Signal type | fluorescence | fluorescence |
| Platform | High throughput fluorescence microscope | High throughput fluorescence microscope |
| Sensitivity | 4/4 | 4/4 |
| Throughput | High | High |
| End-point/real time | End-point | End-point |
| Multiplexing | Yes, for example it can be multiplexed with for example Live/Dead assays | Yes, for example it can be multiplexed with Live/Dead assay |
| Model system | Adherent 2D cell cultures or cytospun suspension cells | Adherent 2D cell cultures or cytospun suspension cells |
Example output
