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Cell cycle analysis by image cytometry

– 2-step cell cycle assay using the NucleoCounter® NC-3000™

Cell cycle and cell division represents the most fundamental and important processes in eukaryotic cells. With the 2-step cell cycle assay using the NucleoCounter® NC-3000™, a full cell cycle profile of mammalian cells can be obtained within 10 minutes.

DAPI fluorescence intensities, which represents individual cell cycle stages, are quantified into histograms and displayed in a user friendly interface.

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Key benefits:

  • Fast and easy measurement of cell cycle phases
  • Single-step acquisition, analysis and data presentation
  • Consistent results even with different users
  • No RNase treatment required
  • No calibration required
  • Superior presentation and management of data

2-step cell cycle assay

 

2 step cell cycle analysis

The 2-step Cell Cycle Assay facilitates detaching, permeabilization, declumping and homogenous staining of the cell population in two simple steps without trypsination, washing and centrifugation. Preparation of samples takes 10 minutes.

Cell cycle of fixed cells assay

 

Fixed cell cycle analysis

The Cell Cycle of Fixed Cells Assay 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.
Application Note (.PDF):

Cell cycle analysis with the NC-3000™

 

Cell Cycle Analysis of Jurkat cells

With the NucleoCounter® NC-3000™ cell cycle analysis has never been faster. Simply add lysis- and staining buffers to cells followed by the addition of a stabilization buffer, load the sample and press run.

A cell cycle profile will be displayed in the accompanying PlotManager software and percentage or number of events in G0/G1, S-phase, G2 or sub-G1 can be identified.

With the FlexiCyte™ software package, the NC-3000™ can even be used for studying cell proliferation with e.g. BrdU or EdU incorporation detected by fluorescently labeled antibodies or Click Chemistry allowing for advanced studies in cell proliferation.

Cell cycle using DAPI

The NC-3000™ Cell Cycle Assays use the nuclear stain, DAPI, to measure DNA content. DAPI binds specifically to double-stranded DNA and, accordingly, there is no requirement for removing RNA prior to DNA content measurement. This is a prerequisite for other dyes commonly used for measurement of cell cycle phases, such as propidium iodide.

Using fluorescence microscopy and image analysis, the NC-3000™ system automates DNA content quantification and hence, measurements of cell cycle stages.

Cell Cycle Assay Princilple - NC-3000
Sample preparations are loaded into either of two types of ChemoMetec slides:

  • 2-chamber NC-Slide A2™ or
  • 8-chamber NC-Slide A8™

Samples are analyzed using the NC-3000™ system where cellular fluorescence is quantified into DNA content histograms. Markers in the displayed histograms can be used to identify cells in different cell cycle stages.

Cell cycle results presented in PlotManager

 

Cell Cycle Analysis - Results presented in Plot Manager

The figure above depicts 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 from the NucleoView™ NC-3000™ software.

Markers in the above displayed 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.

Instruments

Xcyto® 5 NucleoCounter® NC-3000™ NucleoCounter® NC-200™ NucleoCounter® NC-202™ NucleoCounter® SP-100™ NucleoCounter® YC-100™

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