Accurate and Automated cell confluence assessment in microplates TECAN S SPARK 20M MICROPLATE READER WITH INTEGRATED CELL IMAGING SIMPLIFIES CELL CULTURE QC AND SIGNAL NORMALIZATION TO CELL CONFLUENCE
Application Note 2 INTRODUCTION Cell-based assays in microplate formats have become a vital part of life science. Proliferation, cytotoxicity and gene expression studies all use cells as a working tool. Regardless of the research area, cell density analyses and quality checks must be routinely performed to assess the health of the cells, particularly during long-term experiments. In cell culture applications, confluence assessment is an important quality control parameter that is commonly used to estimate the proportion of adherent cells on a growth surface as an indicator of the cell density inside a well or culture flask. Some cell lines behave differently regarding their growth rate or gene expression depending on the degree of confluence. Consistent determination of cell confluence and estimation of cell numbers are therefore important for reproducible assays and accurate interpretation of experimental data. However, visual cell imaging using microscopes is usually a time-consuming and laborious process, especially when multiple samples are being analyzed. A solution for this problem is automated cell confluence assessment. It greatly facilitates the experimental workflow and increases the testing throughput while minimizing experiment-to-experiment variations due to variable starting conditions. Live imaging-based readouts are used to monitor the growth and health of the cells during long-term experiments and offer a reliable quality check for the customer. CONFLUENCE SOFTWARE APP The imaging function is complemented by an intuitive, easy-to-use software application for confluence analyses in SparkControl software, enabling reliable measurements in various plate formats and user-definable patterns (Figure 1). With its comprehensive and user friendly functions it can be used for single- or multi-label confluence measurements in endpoint or kinetic mode, with a measurement time of less than 5 min for an entire 96-well plate with a single image per well. Figure 1: Confluence stripe in SparkControl software. The easy-to-use software stripe allows selection of different areas within the well (see Figure 2). Depending on the assay requirements, the user can either select just one centered picture per well or a full-well picture created from multiple side-by-side images. In addition, the software offers automatic well border detection to compensate for dimensional variations of the microplates. Tecan s Spark platform has an integrated bright-field cell imaging module that enables cell counting and cell viability analyses with an easy-to-use, disposable Cell Chip. This imaging module now also enables label-free and real-time assessment of cell confluence in microplate wells. Based on its bright-field imaging optics, the reader is able to detect cell-covered areas in 6- to 96-well plate formats and calculate the relative confluence ratio based on the analyzed area. The confluence ratios can also be used to normalize any other cell-associated signal (e.g., fluorescence) to the amount of cells. Figure 2: Options for confluence measurement patterns. In the context of this application note, the confluence measurement in the Spark 20M was compared in a longterm experiment to a simultaneous fluorescence-based readout of cell-associated GFP signal as an indicator of cell growth.
Application Note 3 MATERIALS & METHODS A Spark 20M multimode reader was used for the long-term experiment. The instrument s integrated GCM (Gas Control Module) was used and set to 37 C and 5% CO 2. The plate was placed inside Tecan s proprietary Humidity Cassette, which was filled with distilled H 2 O during the readout to provide optimal cell growth conditions. The lid of the Humidity Cassette was automatically removed during each readout using the Spark s patented integrated Lid Lifter TM. RESULTS Figure 3 shows the confluence and fluorescence signals obtained in the long-term experiment (data points shown in 4-h intervals for convenience). Both curves exhibit nearly identical kinetics, showing a steady increase from about 15% confluence (corresponding to about 8,000 RFU) to 50% confluence (about 32,000 RFU). Human squamous epithelial carcinoma cells (A431, ATCC No.1555) stably transfected with GFP were grown to confluence in DMEM high-glucose (Sigma) supplemented with L-glutamine, sodium pyruvate, penicillin/ streptomycin, HEPES and 5% heat-inactivated FCS (PAA Laboratories) at 37 C and 5% CO 2 in a humidified atmosphere. The cells were harvested out of their growth flasks using trypsin/ EDTA and seeded into 96-well tissue culture plates (Greiner, No 655162) at a low initial density to permit unhindered growth over 48 h. In order to achieve highest-possible measurement sensitivity, especially for the fluorescence readout, the experiment was performed in phenol red-free culture medium. Confluence and GFP-based fluorescence signal were recorded in parallel at defined intervals over a period of 48 h. Detailed measurement settings are summarized in Table 1. The microplate was moved to the instrument s incubation position between the individual reads. Figure 3: Dynamics of confluence and fluorescence signals measured in the Spark 20M. The extent of confluence is visualized by a software algorithm that depicts all identified cells in green. The determined confluence value is shown in the upper left corner of the well picture and exported into an interactive Excel worksheet at the end of the measurement. Table 1. Measurement settings Parameter Setting Measurement Kinetic Kinetic duration 48 h Kinetic interval 15 min Meas. Mode 1 Confluence Pattern Whole well Data analysis Activated Settle time 200 ms Meas. mode 2 Fluorescence Intensity Bottom EX wavelength 485 (20) nm EM wavelength 535 (20) nm Flashes 15 (5x3/well, optimal read) Gain optimal (90%) Z position Calculated from well Figure 4: Green overlay highlighting cell-covered areas illustrates continuous increase in cell number.
Application Note 4 In the present experiment, the continuous increase in cell number was apparent when looking at the evaluated pictures with the green overlay for cell-covered areas (Figure 4). To facilitate data handling, export and further evaluation, the measurement results are summarized in an Excel worksheet with active hyperlinks to the recorded picture sets (one bright-field overview and one evaluated picture with the detected confluence value per well). Figure 5 shows the confluence ratio versus the GFP-based fluorescence signal of the cells. Both signals exhibit an increase over time, indicating progressive growth of the cells. Remarkably, the confluence and the fluorescence values show a direct and highly linear correlation, indicated by the excellent correlation coefficient (R 2 value) of 0.997. CONCLUSION The confluence function of the Spark 20M provides an accurate and reliable means for determining the growth status of cells growing in microplate wells. The detected cell confluence directly correlates with the GFP fluorescence signal in a highly accurate and reproducible manner, indicating that the sensitivity of the two readouts is comparable. As the confluence function is label-free, it does not interfere with any other characteristics and properties of the analyzed cells, allowing for the generation of growth curves even with cells that are not transfected with a fluorescent marker like GFP. Furthermore, the distribution of the cells across the well bottom can be easily monitored using the confluence function, enabling new types of applications such as cell migration and repopulation studies with high accuracy and reproducibility. This makes the readout equally well suited for assay optimization and quality control, increasing cell analysis throughput and providing a true walkaway solution for assay automation. Together with its established cell counting and viability functions, the new confluence measurement mode makes the Spark 20M even more versatile for cell-based research. ABBREVIATIONS Figure 5: Correlation of confluence (X axis) and fluorescence (Y axis) signals. DMEM Dulbecco s modified Eagles medium EDTA Ethylenediaminetetraacetic acid EM emission EX excitation FCS Fetal calf serum GFP green fluorescent protein HEPES Hydroxyethyl piperazineethanesulfonic acid RFU relative fluorescent units
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