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Aggregated Cell Count Assay

Precise cell count and viability of highly aggregated cells

  • Determine cell count and viability of aggregated cells in two easy steps
  • User-friendly protocol with pre-defined settings
  • Fast, automated cell analysis
  • One assay for all NucleoCounter® instruments
  • Automated PDF reports

Overview of the aggregated cell count assay

In the Aggregated Cell Count Assay, you lyse and count the cells for the total cell count. For the dead cell count you stain the sample with DAPI before counting.

Aggregated cells such as sphere cultures, iPSCs, and breast cancer cell lines pose great challenges to cell counting. Individual cells superimposed on each other are difficult to distinguish and, therefore, can be challenging for most automated cell counters to assess correctly.

When using trypan blue to stain cells in a hemocytometer or flow-based system using bright-field, counting cells or determining the viability of heavily aggregated cells can be impossible if a clump consists of 5, 10, 20, or even more cells.

The NucleoCounter® instrument counts nuclei instead of whole cells, ensuring clumps do not interfere with the cell counting and viability assessment. When using a carefully developed lysis buffer, our optimized two-step protocol precisely determines cell counts and viability of heavily aggregated cells:

 

  • Step one: All cells are lysed and counted using DAPI, the fluorescent DNA stain
  • Step two: Only dead cells are counted using DAPI. Within just 30 seconds, the NucleoCounter® reports the precise cell count and viability

Assay principle

Upon cell lysis, total and dead cells are counted using the Via2-Cassette™ and NucleoCounter® NC-202™ instrument. As the nuclei only make up a fraction of the total cell area, the NucleoCounter® can reliably distinguish and quantify individual cells that would be difficult, if not impossible, to analyze under bright-field conditions.

If you would like to include further analyses with your cell count and viability analysis, you can stain samples with DAPI and reagents of your choice and analyze them using the NC-slide A2™ and NC-Slide A8™ with the NucleoCounter® NC-3000™.

Cell counting results from the Aggregated Cell Count Assay. Cells have been stained with DAPI and appear blue in the data image.

Results

Comparison of cell counting results from the Aggregated Cell Count Assay between untreated and lysis-treated MCF-7 and CHO-S cells. Untreated cells are more challenging to count than lysed cells.

The images illustrate how the lysis buffer dissociates strongly aggregated MCF-7 cells (top panels) and CHO-S cells grown on microcarriers (bottom panels).

Overall, the same number of total cells are present in the samples. The uniform distribution after lysis ensures a less biased quantification than when counting on a subset aliquot of the sample. Without lysing, there could be a large variation in the cell counting from evaluating sample aliquots containing a considerable amount or very few aggregates or microcarriers. 

The NucleoCounter® instruments have different protocols for different cell types and instruments, so make sure to follow the correct application note for your instrument and sample type.

Publications for reference

  1. C Krabbe, ST Bak, P Jensen et al.: Influence of oxygen tension on dopaminergic differentiation of human fetal stem cells of midbrain and forebrain origin. PLoS One. 2014; 9(5): e96465
  2. P Jackson, K Kling, KA Jensen et al.: Characterization of genotoxic response to 15 multiwalled carbon nanotubes with variable physicochemical properties including surface functionalizations in the FE1-Muta™ mouse lung epithelial cell line. Environ Mol Mutagen. 2014; Volume 56 (2); 183-203.
  3. TG Otsuji, J Bin, A Yoshimura et al.: A 3D sphere culture system containing functional polymers for large-scale human pluripotent stem cell production. Stem Cell Reports. 2014; Vol. 2; 734–745.