Confluency analysis to monitor growth and proliferation
Cell confluence is defined as the percentage of the surface area of a 2D culture that is covered with cells. Commonly confluence assessment is used to determine when cells need to be passaged. Properly timing this moment is essential to maintain cell phenotype and culture quality.
Growth rate monitoring - video example CytoSMART Lux2
Follow the progression of your culture. The video below depicts the growth of C6 cells 120 hours after seeding. The accompanying graph is produced by the confluence detection algorithm in the CytoSMART cloud.
The cell coverage is plotted along the y-axis over the 120 hours culture period. The cell culture progresses from the lagging, to the exponential growth phase, until at nearly a hundred percent coverage a plateau is reached and growth is halted.
Cell area coverage - video example CytoSMART Lux2
See the migration of 3T3 cells over a period of 14 hours at a 15 min imaging interval. Cells are highlighted with a digital mask indicating detection by the CytoSMART confluence algorithm.
whole well analysis - application example CytoSMART Omni
Confluency measurements provide an overview on how cells are distributed throughout sample wells. In the figure bellow JnHEK cells were monitored over a period of 48 hours. Two features are highlighted. The CytoSMART Omni's ability to create bright-field images of large surfaces, while maintaining a detailed view of the cell population. Secondly integrated image analysis algorithms were used to determine confluency values at each time point. The bottom row of images display the confluency mask that is generated after analysis.
The graph displays the confluency in [%] of the top row in the example figure. Imaging and analysis was performed every two hours.
Cell Viability Analysis
Cell viability, growth and cytotoxicity studies can be performed using metabolic activity assays. The overall metabolic activity of the cell is indicated by the enzymatic cleavage of colorimetric or fluorescent substrates.
While these assays are relatively straightforward and cheap, they are dependent on culture conditions and intrinsic metabolic activity of the cell type that is being investigated. Furthermore, depletion of the metabolic substrate can lead to a plateau in the fluorescent signal, making assay output unreliable. To overcome these limitations cell viability could be determined optically using confluency measurements.
In the study described here the performance of confluency measurements to assess cell viability were compared to a metabolic activity assay: cell titer blue. Confluency was visualized using automated bright-field microscopy and subsequently analyzed using image analysis algorithms. Images were collected inside a CO2-incubator, keeping the culture at optimal conditions. For the cell titer blue assay resazurin was added to the medium and incubated for 3 hours. The fluorescent signal was normalized to the control to obtain the relative metabolic activity as a measure of cell viability. The comparison between the methods was performed for two pancreatic cancer cell lines, PACO7 and POCA43.
Cell Culture Monitoring - Lux2
Setting up cell cultures is easy enough. However monitoring your cultures and optimization is time-consuming and cumbersome. Waiting for the ideal confluency, quickly studying effects of various media means taking your cells in and out of the incubator more often than you would like.
Visualizing cell cultures from inside an incubator using a compact microscope that facilitates live cell imaging can overcome these issues. While live cell imaging has been restricted to costly, high-end devices, the CytoSMART Lux2 offers an affordable and easy-to-use alternative for virtually any lab. The CytoSMART Lux2 can be set up in minutes, enabling untrained users to quickly perform their own time-lapse recordings.
Images and videos can be easily accessed and retrieved from the CytoSMART cloud portal. Advanced functions, such as reporting of cell confluency, cell migration analysis and the option to use automatic confluency email alerts, can be applied to inform the user when certain culture conditions are reached (for example, once the cell culture has reached the desired confluency). Hence, the CytoSMART Lux2 can be used in many different ways to facilitate cell culture work and research.
In the following appnote several examples of applications of the CytoSMART Lux2 will be shown.
- Culturing cells in hypoxic conditions
- Standardizing cell culturing conditions
- The effect of confluency on transfection efficiency