Accurate and Reliable Determination of Growth Status of Cells in Microplate Wells

Cell-based assays in microplate formats are increasingly becoming important in the field of life science. Cells are used as a working tool in gene expression, cytotoxicity, and proliferation studies. Irrespective of the research area, it is essential to routinely perform cell density analyses and quality checks to evaluate the cell health, especially during long-term experiments.

Confluence assessment is a key quality control parameter in cell culture applications to calculate the proportion of cells adhered to a growth surface, which is a measure of the cell density within a culture flask or well.

Some cell lines exhibit different growth rate or gene expression based on the degree of confluence. Therefore, consistent estimation of cell numbers and determination of cell confluence are crucial to reproducibly perform assays and accurately interpret experiment data. However, visual cell imaging with microscopes is typically both laborious and time intensive, particularly in analyses involving multiple samples.

Automated cell confluence assessment can address this problem as it simplifies the experimental workflow, improves the testing throughput, and reduces the variations between experiments caused by variable starting conditions.

The cell growth and health during long-term experiments can be monitored using live imaging-based readouts, offering a reliable quality check for users. The built-in bright-field cell imaging module in Tecan’s Spark platform facilitates cell counting and cell viability analyses with a user-friendly, disposable Cell Chip™.

Label-free cell confluence assessment in microplate wells can now be performed in real time using this imaging module. The bright-field imaging optics enables the reader to determine cell-covered areas in 6- to 96-well plate formats.

Based on the area of interest, the relative confluence ratio can then be calculated. Any other cell-associated signal, for instance, fluorescence, to the amount of cells can also be normalized using the confluence ratios.

Confluence software app

An intuitive, user-friendly software application in SparkControl™ software for confluence analyses complements the imaging function, thereby allowing for reliable measurements in a range of plate formats and user-definable patterns (Figure 1).

The extensive and easy-to-use functions of this application enable it to be employed for single- or multi-label confluence measurements in kinetic or endpoint mode, with a measurement time of below 5 minutes for a complete 96-well plate with a single image per well.

Confluence stripe in SparkControl software.

Figure 1. Confluence stripe in SparkControl software.

Different regions in the well can be selected using the user-friendly software stripe (Figure 2). One centered picture per well or a full-well picture generated from multiple side-by-side images can be selected based on the assay requirements.

Moreover, the dimensional differences of the microplates can be compensated for by the software using automatic well border detection. This article compares the confluence measurement in a long-term experiment using the Spark 20M to a concurrent fluorescence-based readout of cell-associated GFP signal as a measure of the cell growth.

Options for confluence measurement patterns.

Figure 2. Options for confluence measurement patterns.

Materials and methods

The long-term experiment used a Spark 20M multimode reader. The integrated Gas Control Module or GCM of the reader was set to 37 °C and 5% CO2 and used. The microplate was introduced into Tecan’s proprietary Humidity Cassette, which was loaded with distilled H2O during the readout to yield optimal cell growth conditions.

The Spark’s patented integrated Lid LifterTM automatically removed the lid of the Humidity Cassette during each readout. Human squamous epithelial carcinoma cells (A431) that were stably transfected with GFP were developed to confluence in DMEM high-glucose (Sigma) that was supplemented with 5% heat-inactivated FCS (PAA Laboratories), HEPES, penicillin/ streptomycin, sodium pyruvate, and L-glutamine in a humidified atmosphere at 37 °C and 5% CO2.

Trypsin/EDTA was used to collect the cells from their growth flasks, and the harvested cells were then seeded into 96-well tissue culture plates (Greiner) at a low initial density to enable unhindered growth over a period of 48 hours.

The experiment was carried out in phenol red-free culture medium to reach the highest-possible measurement sensitivity, for the fluorescence readout in particular. Confluence and GFP-based fluorescence signal were concurrently recorded at defined intervals over 48 hours.

Table 1 lists the detailed measurement settings. The microplate was moved to the incubation position of the instrument between the individual reads.

Table 1. Measurement settings





Kinetic duration

48 hours

Kinetic interval

15 minutes

Meas. Mode 1



Whole well

Data analysis


Settle time

200 ms

Meas. mode 2

Fluorescence Intensity Bottom

EX wavelength

485 (20) nm

EM wavelength

535 (20) nm


15 (5x3/well, optimal read)


optimal (90%)

Z position

Calculated from well


The confluence and fluorescence signals collected in the long-term experiment are plotted in the graph shown in Figure 3. Nearly identical kinetics can be observed in both curves, with a steady increase from 15% confluence (in correspondence with 8,000 RFU) to 50% confluence (corresponding to about 32,000 RFU).

Dynamics of confluence and fluorescence signals measured in the Spark 20M.

Figure 3. Dynamics of confluence and fluorescence signals measured in the Spark 20M.

A software algorithm visualizes the extent of confluence, depicting all identified cells in green. The measured confluence value is depicted in the upper left corner of the well picture.

At the end of the measurement, the measured values are exported into an interactive Excel worksheet. As observed in the evaluated pictures, the cell number increases continuously in this analysis as highlighted by the green overlay for cell-covered areas (Figure 4).

The measurement results are exported into an Excel worksheet with active hyperlinks to the saved picture sets (one bright-field overview and one assessed picture with the measured confluence value per well).

Green overlay highlighting cell-covered areas illustrates continuous increase in cell number.

Figure 4. Green overlay highlighting cell-covered areas illustrates continuous increase in cell number.

The confluence ratio versus the GFP-based fluorescence signal of the cells is shown in Figure 5. An increase is observed for both the signals over time, suggesting progressive cell growth. Notably, a direct and highly linear correlation (R2 = 0.997) is exhibited by the confluence and the fluorescence values.

Correlation of confluence (X axis) and fluorescence (Y axis) signals.

Figure 5. Correlation of confluence (X axis) and fluorescence (Y axis) signals.


From the results, it is evident that the growth status of cells in microplate wells can be accurately and reliably detected using the confluence function of the Spark 20M. The detected cell confluence and the GFP fluorescence signal can be directly correlated in a highly accurate and reproducible manner, implying that the two readouts exhibit comparable sensitivity.

The label-free confluence function prevents interference with any other properties and characteristics of the cells of interest and therefore, growth curves can be generated even for cells that are not transfected with a fluorescent marker such as GFP.

In addition, the confluence function facilitates the monitoring of the cell distribution across the well bottom, allowing for new applications such as cell repopulation and migration studies with very high accuracy and reproducibility.

This capability makes the readout equally suitable for assay optimization and quality control, improving the throughput of the cell analysis and offering a true walkaway system for assay automation. The Spark 20M is a versatile system for cell-based research owing to the combination of capabilities, such as proven cell counting and viability functions and the new confluence measurement mode.


About Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world

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Last updated: Feb 11, 2020 at 4:33 PM


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