Confluence analysis to monitor cell growth and proliferation

Cell confluence is defined as the percentage of the surface area of two-dimensional (2D) culture that is covered with cells. Commonly confluence assessment is used to determine the point when cells need to be passaged. Properly timing this moment is essential to maintaining cell phenotype and culture quality.


Researchers can easily monitor and analyze cell proliferation with these CytoSMART devices: full-plate scanner Omni, fluorescence cell imager Lux3 FL, mini live-cell imagers Lux2 and Lux3 BR.


In the example below, C6 glioma cells were imaged for a total period of 5 days (120 hours) using the brightfield imaging system, the CytoSMART Lux2 (Figure 1). The integrated confluence detection algorithm automatically generates a cell proliferation curve (Figure 2). Here, the cell coverage is represented by the y-axis, whereas the overall imaging time is plotted on the x-axis. Over the course of a 5-day period, the culture progresses from the lagging to the exponential growth phase, until at nearly 100% coverage a plateau is reached and growth is halted.

Figure 1 | Time-lapse recording of C6 cells proliferation. The C6 glioma cell line was seeded and imaged using the CytoSMART Lux2 for 5 days, generating a time-lapse video that is automatically uploaded to the CytoSMART Cloud.

Proliferation curve of C6 cells over a 5-day period

Figure 2 | Proliferation curve of C6 cells over a 5-day period. The graph was generated automatically using the CytoSMART confluence detection algorithm.

The CytoSMART Omni: whole-well analysis of cell confluence

In addition, the confluence detection algorithm of the brightfield imager, the CytoSMART Omni provides an overview of cell distribution throughout multiple wells of a tested multi-well plate (Figure 3). Here, JnHEK cells were monitored over a 48-hour period, with imaging and analysis being performed every 2 hours. The confluence mask (green overlay) is generated automatically to highlight the position of cells, as detected by the CytoSMART confluence algorithm.

cell confluence

Figure 3 | The CytoSMART confluence detection algorithm. The integrated confluence detection algorithm was applied to brightfield images (top row) acquired using the CytoSMART Omni. The algorithm highlighted (in green) the distribution of JnHEK cells within individual wells of a 96-well plate (bottom row).

The CytoSMART Lux3 FL: fluorescence-based analysis of cell confluence

The fluorescence live-cell imager, the CytoSMART Lux3 FL, can assess cell confluence using both, brightfield and fluorescence images. The robust image analysis algorithm enables accurate and reliable analysis of cell proliferation over time.

Here, HepG2 human hepatoma cells were transduced in suspension using CellLight Nucleus-RFP BacMam 2.0 and CellLight Actin-GFP BacMam 2.0 reagents for detection of nuclei and filamentous actin, respectively. Subsequently, the cells were monitored using the CytoSMART Lux3 FL with an imaging interval of 30 min (Figure 4; Figure 5).

Fluorescence cell confluence

Figure 4 | Automated cell confluence and confluence ratios plots generated by the Lux3 FL. Following the transduction with BacMam Nucleus-RFP (nuclei) and BacMam Actin-GFP (actin filaments), HepG2 cells were imaged using the CytoSMART Lux3 FL and the automated data report was generated. (A) In the cell coverage plot, the total cell coverage is indicated by the blue line, whereas the red and green lines indicate the percentage of red fluorescence and the green fluorescence coverage, respectively. (B) In the fluorescence coverage ratios plot, the green line indicates the percentage of coverage ratio of green fluorescent objects, whereas the red line represents the coverage ratio of red fluorescent objects.

Figure 5 | Time-lapse video of transduced HepG2 cells. HepG2 cells, transduced with BacMam Nucleus-RFP (nuclei) and BacMam Actin-RFP (actin filaments), were imaged using the CytoSMART Lux3 FL. The fluorescence confluence detection algorithm highlights red fluorescence areas (BacMam Nucleus-RFP; cell nuclei) and green fluorescence areas (BacMam Actin-GFP; actin filaments).

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