Drug discovery solutions

Cell cycle analysis using multiparameter flow cytometry

Cell cycle phases

Cell cycle phases
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The cell cycle is a strictly regulated process that is responsible for appropriate cellular growth, development, and differentiation. It combines DNA replication with chromosomal segregation to ensure equal distribution of duplicated genetic material in the daughter cells. 

Cell cycle phases are divided into four sequential stages that progress from quiescence (G0 phase) to proliferation (G1, S, G2, and M phases), and back to G0 phase. The deregulation of this process is considered a hallmark of tumor cell development. 

Therefore, both academic and industrial drug discovery programs have strongly focused on the development of drugs targeting cell cycle phases. Using advanced flow cytometry researchers can rapidly and precisely determine different phases of the cell cycle.

DNA content

Cell cycle results from flow cytometry-based DNA content analysis displayed as a histogram
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Traditionally, cell cycle phases are analyzed by evaluation of a DNA histogram generated by flow cytometry. 

This technique is fast and reproducible and can, for a well-defined cell population, give an approximation of the number of cells in G0/G1, S, and G2/M. 

However, G0 and G2 cells cannot be separated from G1 and M cells, respectively. This limitation can partly be overcome by acridine orange staining giving discrimination of G0 from G1

Furthermore, histogram analysis of tumor samples with aneuploid populations can be difficult and sometimes impossible.

In a flow cytometer cell cycle analysis based on DNA content measurement is usually analyzed on a linear scale since the differences in fluorescence are usually small. Flow cytometry software programs offer algorithms to accurately estimate the cell cycle phases.

Watch a video tutorial on DNA content analysis.

Proliferation-associated protein expression 

Different proliferation- and cell cycle-related antigens have been described, allowing for the evaluation of cell kinetic parameters by detecting the expression of these antigens.

Identification of S-phase cells by detection of PCNA
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Proliferating cell nuclear antigen (PCNA) cyclin is expressed mainly during S phase and acts as a scaffold to recruit proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics. 

PCNA expression has been found to correlate with tumorigenesis, mainly with the degree of malignancy, vascular infiltration, distant metastasis, and survival. This antigen has been described as a biomarker of colorectal adenocarcinoma.


Available products:

Anti-PCNA antibodies, human, mouse, rat

Identification of proliferating cells by detection of the Ki-67 marker
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Ki-67 is a nuclear antigen and it is expressed in actively cycling cells (G1, S, G2 and M phase) but not in resting G0 cells. Ki-67 is considered to be a good marker to identify the mitotic index and the fraction of dividing cells. 

The level of Ki-67 expression is of great prognostic significance in carcinomas of the prostate, breast, and liver, as well as in malignant melanoma, malignant lymphoma, and lung cancer.


Available products:

Anti-Ki-67 antibodies, human and mouse

Cell cycle-associated protein expression

Cyclins are a family of proteins that control the progression of cells through the cell cycle by activating cyclin-dependent kinase (CDK) enzymes. The most important cyclins involved in cell cycle processes are listed below.

ProteinFunctionInhibitory protein
Cyclin AControl of S phase in complex with Cdk1 or Cdk2Cdk1 or Cdk2
Cyclin BControl of G2/M phase in complex with Cdk1Cdk1
Cyclin DControl of G1 phase in complex with Cdk4 or Cdk6; Regulation of Rb/E2F transcriptionCdk4 or Cdk6
Cyclin EControl of G1-S phase in complex with Cdk2; Regulation of Rb/E2F transcriptionCdk2
Cyclin HCdk-activating kinase and RNAPII transcription in complex with Cdk7Cdk7

Phosphorylated Histone H3

Identification of M phase cells by detection of phosphorylated histone H3
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Histone H3 becomes phosphorylated only during M phase of the cell cycle. 

Thus, histone H3 pSer28 can be used as a specific marker for M-phase cells and combined with other proliferation tools to further segment cell cycle compartments.


Available products:

Anti Histone H3 pS28 antibodies, human and mouse

Check out our resources and technologies for optimal multiparameter flow analyses