Cell cycle analysis using multiparameter flow cytometry

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.

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.

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

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

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