FlexiCyte™ Assay

Fully Flexible Data Visualization and Analysis

  • Advanced cell analysis of a broad range of fluorescent biomarkers and proteins
  • Compliant: 21 CFR Part 11/GMP-ready
  • Adjustable analysis settings before and after data acquisition
  • Integrated image analysis algorithm
  • Data visualization and validation during acquisition

Overview of the FlexiCyte™ Assay

The FlexiCyte™ Assay for the NucleoCounter® NC-3000™ advanced image analyzer, enables users to perform detailed cell analysis of a broad range of biomarkers and fluorescent proteins in mammalian cells. The ‘Protocol Adaptation Wizard’ guides the user through a selection of optimal settings.

FlexiCyte™ allows you to build a custom assay using a combination of LEDs from UV to far-red matched with carefully selected emission filters contained within the NucleoCounter® NC-3000™. This allows for simultaneous detection of numerous fluorescent antibodies and proteins.

The ‘Protocol Adaptation Wizard’ guides you through the selection of optimal settings. After image acquisition, data is presented as scatter plots and histograms in the Plot Manager, along with the fluorescent image. Detailed data analysis can then be performed linking individual events between images and plots for full understanding of co-localization of fluorescent events and cell (sub-)populations.

Assay Principle

Samples are stained with fluorophores specified by the user. Note that the default FlexiCyte™ protocol requires counterstaining of the sample with either DAPI or Hoechst-33342 in addition to the user specified staining. DAPI and Hoechst-33342 stain the nucleus and are used for detection (i.e. masking) of the total cell population. Hoechst-33342 stains both living and dead cells and is used for labeling samples with non-fixed cells. DAPI exclusively stains dead cells and is used for labeling cells fixed with either alcohols or aldehydes.

For staining of cellular markers, we recommend using Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 647. These dyes are bright and photostable and have minimal spectral overlap. Avoid using dyes that are prone to photobleaching, such as FITC, PE, APC and PerCP.

For full information and recommendations, please refer to the Application Note: FlexiCyte™ Protocol for the NucleoCounter® NC-3000™ system .

Results Presented in Plot Manager

Use the Plot Manager to analyze the events acquired by the NucleoView™ software with the NucleoCounter® NC-3000™. Draw polygons, quadrants, and markers in the plots to count and gate sub-populations for detailed data analysis and verification of staining events.


  • Instrument: NucleoCounter® NC-3000™
  • Cell Type: Mammalian Cells
  • Assay: Cell Count & Viability Assay, FlexiCyte™ Assay
  • Research area: Oncology

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