Counting aggregated cells
Using the NucleoCounter® instruments
NucleoCounter® instruments use fluorescent dyes to stain nuclei of cells in aggregates
Light microscopy highlights the contour of objects based on their shade pattern (Figure 1). 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 and DAPI – all fluorescent dyes with affinity for DNA – 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 (Figure 1). Moreover, cellular debris and other artifacts that otherwise would be visible under light microscopy become undetectable under fluorescence microscopy.
Figure 1: Light versus fluorescence microscopy for counting cells in aggregates. Artifacts visible by light microscopy techniques (Light Microscopy), such as bright field, are not visible when using fluorescence filters. Also, nuclei fluorescing with Acridine Orange (Fluorescence Nuclear Staining) gives an outline that is smaller than whole cells and with enhanced contrast, allowing better identification of individual cells in aggregates. Images in this figure are from the same cells/area acquired using a proprietary microscopy system configured with light microscopy and fluorescence filters.
NucleoView™ singles out individual cells within smaller aggregates
Nucleoview™ is the software part of the NucleoCounter® system. It employs a powerful algorithm to accurately identify and analyze individual cells within smaller aggregates. Intensity peaks and corresponding slopes are used to segment objects relative to background intensity levels. Cells that are close together will still present identifiable discrete peaks, even though their corresponding slopes might overlap (Figure 2). Intensity values of segmented cells are represented graphically for better visualization and validation of data.
Figure 2: Automated image analysis with the NucleoView™ software. Images acquired by the NucleoCounter® instrument are instantly processed by Nucleoview™ software’s powerful algorithm. Intensity peaks and corresponding slopes are used to segment individual cells, including those within aggregates. The data is converted plots for easier processing by the user.
NucleoCounter® instruments give a numerical estimation of cell aggregation
Counting heavily aggregated cells or clumps with NucleoCounter®
Certain cell types (such as MCF-7, HepG2, iPSC, etc), cells grown in spheres or microcarriers and tissue samples will tend to form large clumps that are resilient to enzymatic or mechanical separation methods. Individual cells in such large aggregates will tend to be superimposed, reducing accuracy and precision of the cell count. NucleoView™ will give the percentage of cells in aggregates with more than five cells to flag samples where cell aggregation could affect the cell count. The dedicated Aggregated Cell Assay is recommended for such samples. In the Aggregated Cell Assay, the total cell count derives from a separate lyzed aliquot of the sample (Figure 3). 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 (Figure 3).
Figure 3: Schematic representation of the “Viability and Cell Count – Aggregated Cells Assay” protocol.