Apoptosis

Cell Cycle and Cell Proliferation: An Overview

To help researchers better understand the fundamental cellular mechanisms involved in immunity, inflammation, hematopoiesis, neoplasia, and other biological responses, BD Biosciences offers a range of tools including antibodies, kits, and systems to measure proliferative responses.


Using flow cytometry, immunofluorescence, or immunohistochemistry, researchers can quickly and accurately determine the cell cycle status or tissue localization of individual cells within proliferating populations. These tools include:

  • BD Biosciences reagents and BD Cycletest™ Plus reagent kit for the analysis of cellular DNA content
  • DNA dyes, propidium iodide (PI), 7-aminoactinomycin D (7-AAD)
  • Antibodies against cyclins, retinoblastoma, and phosphorylated histone H3

In adaptive immunity, specific T and B lymphocytes undergo clonal expansion (division, proliferation, and differentiation) in response to foreign antigenic stimulation. Cell growth, replication, and division in eukaryotic cells occur according to a highly controlled series of events called the cell cycle.

Cell cycle phases

The Cell Cycle

The cell cycle has two major phases: interphase, the phase between mitotic events, and the mitotic phase, where the mother cell divides into two genetically identical daughter cells. Interphase has three distinct, successive stages. During the first stage called G1, cells "monitor" their environment, and when the requisite signals are received, the cells synthesize RNA and proteins to induce growth. When conditions are right, cells enter the S stage of the cell cycle and "commit" to DNA synthesis and replicate their chromosomal DNA. Finally, in the G2 phase, cells continue to grow and prepare for mitosis.

Cell cycle phases

Analysis of Cellular DNA Content

BD Biosciences offers a wide variety of reagents to study the cell cycle. Reagents include DNA dyes such as propidium iodide (PI) and 7-aminoactinomycin D (7-AAD). In addition, the BD Cycletest Plus reagent kit includes PI and other reagents to degrade proteins and RNA to allow more precise DNA measurement. The samples are subsequently analyzed using flow cytometry to assess ploidy, identify abnormal DNA stemlines, and estimate the DNA index (DI) and cell cycle phase distributions of stemlines. During the cell cycle phases, DNA levels change, facilitating the use of DNA dyes such as 7-AAD to generate characteristic cellular DNA content profiles (see the figure below). As cells go through the phases of the cell cycle, proteins such as histone H3 Ser28 become modified or change in expression. To facilitate DNA replication the histone is modified, opening the chromatin to allow entry of replication machinery. To further support the study of cell cycle, BD Biosciences carries antibodies to these proteins to use for imaging or flow cytometry applications.

CD4 enriched mouse splenocytes were cultured with anti-CD3/CD28
CD4 enriched mouse splenocytes were cultured with anti-CD3/CD28, IL-2, and IL-4 for 6 days. Cells were harvested and treated with 10 ng/mL IL-2+1 µg/mL colcemid for 4 (left) or 24 (right) hours and stained with the BD Cycletest™ Plus DNA reagent kit.
Cell cycle analysis of a population stained for incorporated BrdU and total DNA levels (7-AAD).
Cell cycle analysis of a population stained for incorporated BrdU and total DNA levels (7-AAD).
Human PBMCs were stimulated with anti-CD3/CD28 for 48 hours and re-stimulated with PMA+ionomycin for 4 hours, and BrdU was added for the final 1 hour. Cells were then harvested and stained using the BrdU staining protocol.

 
Measures Reagents Mechanism Technology Sample Types
DNA Propidium Iodide (PI), 7-aminoactinomycin D (7-AAD) Interaction into DNA double strands Flow cytometry Fixed, permeabilized, and for live/dead discrimination in intact cells
Cell Dyes BD Horizon™ violet proliferation dye 450 (VPD450) Diffuses into live cells and is hydrolyzed by intracellular non-specific esterases to become fluorescent products. Flow cytometry Live proliferating cells
Newly Synthesized DNA BrdU and antibodies to BrdU Bromodeoxyuridine replaces thymidine (T) in dividing DNA. It is then detected by antibodies to BrdU. Flow cytometry, cell imaging, immunohistochemistry Fixed and permeabilized cells, treated tissues (cell imaging, immunohistochemistry only)
Protein Level Antibodies to Ki67, PCNA Levels increase as a result of proliferation. Flow cytometry, bioimaging, immunohistochemistry, Western blot Fixed cells, tissues, and extracts
Protein Level Antibodies to cyclins, retinoblastoma (Rb), other cell cycle markers Levels go up and down at different stages of the cell cycle. Flow cytometry, bioimaging, immunohistochemistry, Western blot Fixed cells, tissues, and extracts
Protein Modification Antibodies to phosphorylated histone H3, cyclin dependent kinases (cdk) Proteins become phosphorylated as a result of proliferation or changes to the cell cycle. Flow cytometry, bioimaging, immunohistochemistry, Western blot  
BD™ CBA (for quantitative detection) Fixed cells, tissues, and extracts