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Application Notes

Cell Counting–Measuring the Number of Cells in Spheroids

Confocal observation combined with the cell imaging analysis system enables users to count the number of cells in spheroids using cross-sectional spheroid images.


Knowing the number of cells is one of the most important parameters in assays using cells, and this is also the case during the analysis of spheroids. Cell counting with a hemocytometer requires a cell suspension that is prepared using a spheroid dissolution with trypsin. If this is done, however, the features of spheroids originating in their 3D structure are lost. In this study, nondestructive cell counting for spheroids was performed as follows: the number of cells in cross-sectional images of spheroids were measured through confocal observation combined with the use of the cell analysis system. The results were compared to those obtained using a hemocytometer.


Preparation of samples

A cell suspension of HeLa, or HT-29, was seeded into PrimeSurface® 96U plate (SUMITOMO BAKELITE CO., LTD) at 500 cells/well. Each spheroid was harvested 48 hours after the start of cell culture. One half of every spheroid was fixed in 4% formalin followed by staining with Hoechst 33342 and transparentizing1 overnight. The remaining part of every spheroid was treated with trypsin overnight to prepare the cell suspension.

1) Susaki et.al. Cell. 2014 Apr 24;157(3):726-39. doi: 10.1016/j.cell.2014.03.042. Epub 2014 Apr 17.

Cell counting with the cell analysis system and hemocytometer

Fluorescent images of the above-mentioned spheroids were obtained using the system. (Ex: 405 nm, Em: 488 nm). To count the cells, the system acquires cross-sectional images of spheroids every 20 μm and then identifies and counts the visible cells (Figure 1*). Next, the number of nuclei are added together to estimate the total number of cells within the entire spheroid (system counting). The results were compared to those obtained using a hemocytometer (manual counting), as shown in Figure 2 (bars: SD, n=6). These results showed that the system enables cell counting for spheroids while maintaining their structural integrity and without having to dissolve the spheroids using trypsin.

Figure 1: (a)(b)(c)

Figure 1*:
(a) Image of cell identification in a spheroid
(b) Image of cell identification in a spheroid displayed in pseudo colors
(c) Image of cell identification at the Z position in a spheroid

Figure 2: Numbers of cells in spheroids measured by the system or hemocytometer

Figure 2:
Numbers of cells in spheroids measured by the system or hemocytometer

PrimeSurface is a registered trademark of Sumitomo Bakelite Co., Ltd.

Olympus is a registered trademark, and NoviSight and Insightful Analysis, Intelligent Answers are trademarks of Olympus Corporation.

*Although it became one of the most important cell lines in medical research, it’s imperative that we recognize Henrietta Lacks’ contribution to science happened without her consent. This injustice, while leading to key discoveries in immunology, infectious disease, and cancer, also raised important conversations about privacy, ethics, and consent in medicine.
To learn more about the life of Henrietta Lacks and her contribution to modern medicine, click here.

Products related to this application

3D Cell Analysis Software


  • 3D image recognition from a whole structure to sub-cellular
  • Statistical analysis
  • Variety of default assays

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