Cell Cycle

The cell cycle refers to the whole process of a cell undergoing from the completion of one division to the end of the next, and the cell cycle is divided into two phases, interphase and mitosis.

Interphase (interphase) is divided into three phases: Pre DNA synthesis (G1 phase), DNA synthesis (S phase), and post DNA synthesis (G2 phase)

The M phase (mitosis), the cell division phase, is divided into: prophase, metaphase, anaphase, and telophase.

The regulation of the cell cycle is mainly achieved by the arrest in the G1 phase. G0 phase refers to the state in which cells are in the arrest. Cells are divided in M phase, some can continue to divide for cycle cycling, and some jump into G0 phase to arrest. The G0 phase is a phase away from the cell cycling which temporarily stops dividing. But upon certain stimulation, the cell can again enter the cell cycle, synthesize DNA and undergo division.

The G0 phase is characterized by: ① unstimulated G0 cells, where the potential for DNA synthesis and cell division remains, and ② when G0 cells are stimulated to proliferate, they can synthesize DNA and undergo cell division.

Technical details

Principle of cell cycle detection by PI staining and flow cytometry:

Propidium iodide (PI) is a fluorescent dye that can be integrated into and bound to base pairs of double-stranded DNA and RNA with no base specificity. PI can produce fluorescence after binding to double-stranded DNA, and the fluorescence intensity and the content of double-stranded DNA are directly proportional. After the DNA inside the cells is stained with PI, the cells can be measured by flow cytometry for DNA content, and then, depending on the distribution of DNA content, analysis of the cell cycle can be performed.

Due to the different DNA content in each phase of the cell cycle, generally, normal cells have a DNA content (2n) of diploid cells in the G1 / G0 phase, and in G2 / M phase cells have a DNA content (4N) of tetraploid cells, and in S phase, the DNA content of the cells is between diploid and tetraploid. PI can bind to DNA, and its fluorescence intensity directly reflects the intracellular DNA content. Therefore, when the intracellular DNA content is examined by flow cytometry combined with PI staining, each phase of the cell cycle can be distinguished into G1 / G0, S, and G2 / M phases. The obtained flow histograms corresponding to each cell cycle can be analyzed by special software and therefore the cell% on each cell cycle can be calculated.

Case study

Reference
B,C. Xie, Kewei et al. “Yes-associated protein regulates podocyte cell cycle re-entry and dedifferentiation in adriamycin-induced nephropathy.” Cell death & disease vol. 10,12 915. 4 Dec. 2019, doi:10.1038/s41419-019-2139-3

Service turnaround and deliverables

Service turnaround

5~10 business days, depending on cell growth and experimental design

Customer provides

cell lines, drugs, drug treatments and experimental conditions parameter settings

Deliverables

raw data, analytical results, experimental reports

Other methods for cell cycle detection

Principle		Features
BrdU assay	After BrdU (5-bromodeoxyuridine) is added to the culture medium, it can be used as the raw material of cell DNA replication, replacing thymidine to integrate into newly synthesized DNA of cells that enter the S phase (DNA synthesis phase). The antibody used to detect BrdU only recognizes the BrdU which is integrated into the single strand of DNA, and PI staining can be used for undenatured double-stranded DNA. Sustained BrdU integration can be used to determine and analyze cells that are in an active state of DNA synthesis, distinguished from those that are in arrest, and thereby used to analysis of the proportion of cells that proliferate; On the other hand, the addition of BrdU at different time points allows the analysis of cell cycle kinetics.	BrdU can integrate into the DNA of S-phase cells and stably exist in daughter cells through DNA replication; BrdU causes irreversible damage to the organism and please be cautious while using.
7-AAD assay	7-AAD（7-amino-actinomycin D) is a nucleic acid dye that is unable to pass through the normal plasma membrane. Along with cell apoptosis and cell death, the permeability of the plasma membrane to 7-AAD increases, and 7-AAD can emit a bright red fluorescence upon excitation with an appropriate wavelength of excitation light. Cells could be classified into three populations by the strength of 7-AAD labeling of DNA: 7-AAD-strong as dead cells, 7-AAD-weak as apoptotic cells, and 7-AAD-moderate as normal viable cells. The DNA content in G0 / G1, S, and G2 / M phases can be detected by flow cytometry.	7-AAD has similar fluorescence characteristics as PI, but its emission spectrum is narrower than that of PI and less interference with other detection channels, making it an ideal alternative to PI in multiplex fluorescence staining. And 7-AAD can be used in combination with several 488 nm exciting fluorescent dyes, such as FITC (fluorescein isothiocyanate), PE (Phycoerythrin), APC (Allophycocyanin), etc.