Cell count & viability of aggregated cells

Fluorescent dyes stain nuclei of cells in aggregates, microcarriers & spheroids

NucleoCounter® instruments offer robust and reliable solutions for counting cells in clumps and aggregates. They use fluorescent dyes to stain cell nuclei and employ a powerful algorithm to segment single nuclei within each smaller aggregate.

For the more heavily clumped samples, a numerical estimation of the level of cell aggregation enables the operator to make an informed decision on whether to switch to the dedicated Aggregated Cell Assay, which disassembles cells with a lysis step. This dedicated assay is also used for counting cells grown in microcarriers or spheroids.

Light vs. fluorescence microscopy for counting cells in aggregates

The same cells seen in a fluorescence-based image (green) and viewed through a light microscope (grey). Cell aggregates are easier to count in the fluorescence-based image, and debris is excluded.
Light microscopy highlights the contour of objects based on their shade pattern. Counting cells using conventional light microscopy methods can lead to the inclusion of contaminations and cellular debris visually indistinguishable from cells. More importantly, when the outer contour of adjacent cells overlaps, as in the case of cell clumps, it can be nearly impossible to accurately identify individual cells.

NucleoCounter® instruments exploit acridine orange (AO) and DAPI, both fluorescent dyes with DNA affinity, to label cellular nuclei with great specificity and generate high-contrast signals. Nuclei are smaller than whole cells and hence better to segment when cells form small aggregates. Moreover, cellular debris and other artefacts that would otherwise be visible under light microscopy, become undetectable under fluorescence microscopy.

Artefacts visible under light microscopy techniques, such as bright-field, are not visible when using fluorescence filters. AO staining results in an enhanced signal from the nuclei, enabling individual cells in aggregates to be identified more easily.

Principle of automated image analysis with NucleoCounter® Software

NC-View™ and NucleoView™ are the software platforms for the NucleoCounter® NC-202™ and NucleoCounter® NC-3000™ instruments, respectively. The software employs a powerful algorithm to accurately identify and analyze individual cells within smaller aggregates.
Cells that are close together will still present identifiable discrete peaks, even though their corresponding slopes might overlap.

Images acquired by the NucleoCounter® instrument are instantly processed by its software’s powerful algorithm. Intensity peaks and slopes are used to segment individual cells, including those within aggregates.

The data is shown as fluorescent images to enable the user to identify and count live and dead cells. With the NucleoCounter® NC-3000™, data is also shown as histogram plots with adjustable gate settings for deeper analysis. This makes it possible for the user to trace back to individual cells and cell populations.

In the NucleoCounter® software, cells are mapped, segmented, and represented in a scatter plot.

NucleoCounter® instruments give a numerical estimation of cell aggregation

The NucleoCounter® system is superior in segmenting cells within small aggregates of less than five cells. Samples with larger aggregates tend to have cells overlapping each other, rendering these samples very hard to analyze. In fact, manual and automated cell counting relying on light microscopy techniques such as bright-field microscopy and phase contrast, fall short in their ability to estimate levels of aggregation in a sample.

The NucleoCounter® operator gets an estimate of the percentage of aggregation in the sample; from this, they can make an informed decision on whether to choose the dedicated Aggregated Cell Count Assay, which enables them to accurately assess the cell count.

Counting heavily aggregated cells or clumps

Tissue samples and certain cell types grown in spheres or on microcarriers, such as MCF-7, HepG2 and iPSC, tend to form large clumps that are resilient to enzymatic or mechanical separation methods. Individual cells in such large aggregates are likely to be superimposed, reducing the accuracy and precision of a cell count.

The NC-View™ and NucleoView™ software give the percentage of cells in aggregates of more than five cells. This flags samples where cell aggregation occurs and as a result, could affect the cell count. The dedicated Aggregated Cell Count Assay is recommended for such samples. In this assay, the total cell count derives from a separate lyzed aliquot of the sample. The lysis process disrupts the cell membrane, dismantling clumps and releasing the nuclei to be stained into solution. Because dead cells are typically less abundant and tend to detach from aggregates, the dead cell count can still be based on the non-lyzed aliquot.

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.


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