Cell cycle assay

Analyze fixed or live cells in two simple steps using the cell cycle assay

  • Fast and easy measurement of cell cycle phases
  • Single-step acquisition, analysis, and data presentation
  • Standardized results – even between different users
  • No RNase treatment required
  • No calibration required
  • Clear data presentation and management
  • Export data in FCS/ACS formats

Overview of the cell cycle assay

In the 2-Step Cell Cycle Assay, the cells are lysed, stained with DAPI, stabilized, loaded into a cell counting slide, and counted with the NucleoCounter® NC-3000™. In the Fixed Cell Assay, the cells are fixed, stained with DAPI, and counted using the NC-3000™.

Cell cycle and cell division represent the most fundamental and important processes in eukaryotic cells. With the Cell Cycle Assay, DAPI fluorescence intensities, representative of individual cell cycle phases, are quantified and displayed in a user-friendly interface.

The 2-Step Cell Cycle Assay (upper flow) facilitates detaching, permeabilization, de-clumping and homogenous staining of the cell population in two simple steps without trypsinization, washing or centrifugation. The Cell Cycle of Fixed Cells Assay (lower flow) enables cell storage for several weeks. Thus, after ethanol fixation, the cells can be retained in cold storage for a much longer period before sample analysis.

With the NucleoCounter® NC-3000™ cell cycle analysis, simply prepare your sample by lysis or fixation, then add staining buffers, load the sample, and press run. After the one-minute data acquisition and analysis is complete, the cell cycle profile data and percentage or number of events in G0/G1, S, G2 or sub-G1 phases displays in NucleoView™ software’s Plot Manager feature.

With the FlexiCyte™ software package, the NC-3000™ can even be used for studying cell proliferation using BrdU- or EdU-incorporation. When detected by fluorescently labeled antibodies or Click Chemistry, these allow for advanced studies of cell proliferation.

Assay principle

The Cell Cycle Assay uses the nuclear stain DAPI to measure DNA content. DAPI binds specifically to double-stranded DNA. Consequently, there is no requirement to remove RNA prior to DNA content measurements. This is a prerequisite for other dyes commonly used for measurement of cell cycle phases, such as propidium iodide (PI).

Diagram of the cell cycle (left) and diagrams showing the distribution of cells in the cell cycle phases under different conditions (right).

Using fluorescence microscopy and image analysis, DNA content quantification and measurements of cell cycle phases are automated.

Sample preparations can be loaded into either of two slide types:

Samples are analyzed using the NucleoCounter® NC-3000™ advanced image cytometer where cellular fluorescence is quantified into histograms displaying the DNA content quantification. Markers in the displayed histograms, M1 – M4, can be used to identify cells in different cell cycle stages: sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase, respectively.

Results presented in plot manager

The histogram plots depict U2OS cells grown in absence (upper row) or presence (lower row) of etoposide. DNA content was measured using the 2-Step Cell Cycle Assay in the NC-3000™. Scatter plots, histograms and tables were obtained using NucleoView™ NC-3000™ software.

Markers in the histograms were used to demarcate cells in the different cell cycle phases. The colored histogram is a merge of untreated (blue line) and etoposide treated (red line) samples. G0/G1 indicated by the M1 marker, S phase indicated by M2, and the G2/M phase cells indicated by M3.

Image of processed data from the Cell Cycle Assay in the NucleoView™ software. Histograms and scatter plots show the DNA content of the cells.