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Automated image-based direct cell counting solutions

Accurate cell counting is a critical step across a spectrum of cell biology applications. Direct cell counting uses methods that identify and enumerate individual cells, as opposed to indirect methods that estimate population sizes using biochemical methods. Automating the process of direct cell counting improves accuracy and consistency while simultaneously reducing experimental burden. Accurate cell numbers are essential for establishing and optimizing cell-based assays, normalizing data across samples, and conducting cell proliferation assays.

The Agilent BioTek Lionheart automated microscope and Agilent BioTek Cytation cell imaging multi-mode readers are flexible systems that can use both label-free and fluorescence imaging methods for cell count determination. Combined with powerful Agilent BioTek Gen5 software image analysis tools, these systems deliver easy-to-use, robust automated cell counting solutions.

High Contrast Cell Counting – Label-Free Direct Cell Counting

cell counting

Label-free direct cell counting is an effective method to achieve the accuracy and sensitivity of direct cell counting without the limitations associated with fluorescent labels.

Agilent BioTek’s high contrast (HC) cell counting technique uses modified brightfield illumination to generate a distinct bright signal corresponding to each cell.  These HC cell signals are readily identified and counted using Gen5 image analysis tools.  HC cell counts are comparable to counts derived using fluorescent nuclear labels across a range of cell densities.

Kinetic Cell Proliferation using direct, label-free Cell Counting

cell counting

Accurate evaluation of cells populations is essential for proliferation assays. Label-free cell counting methods can streamline workflows and minimize cellular manipulation. Label-free methods also increase multiplexing capacity by liberating channels for fluorescence analysis.

Agilent BioTek imaging systems combine label-free cell counting and integrated environmental controls ideal for live-cell proliferation assays. Built-in analysis enables multiple methods to determine total populations through direct cell counting and area confluence.

Fluorescent Cell Counting

Fluorescent labelling is a widely used strategy to directly count cell numbers during image-based cell investigations. Fluorescent nuclear labels enable direct cell counts of cell types that tend to cluster or present challenging morphologies in label-free images.

Agilent BioTek imaging systems support cell quantification across a spectrum of fluorescence cellular and nuclear markers. Gen5 software image analysis tools automatically identify, mask and count cells for easy and robust assessment of proliferation.

cell counting

Advanced Cell Counting Tools - Subpopulation Analysis

Enhance your analysis with advanced cell counting subpopulation tools. Gen5 software provides a comprehensive and customizable feature list to capture the relevant details of cellular phenotypes. Use characteristics such as fluorescence intensity or cell size to determine statistically-derived thresholds for subpopulation analysis.

Achieve automated and statistically-driven determinations of the number of cells within the population responding to treatment.

Data Normalization using Automated Cell Counts

Cell counting provides a straightforward method for normalizing results across sample conditions.

Using Agilent BioTek fluorescence imaging and cellular analysis tools, sample counts of nuclear stained cells are used to normalize Agilent Seahorse XF metabolic data across cell densities. 

Data normalization to cell counts is essential for accurate and reliable determination of cellular readouts.

Automated Cell Counts


The last decade has seen an explosion in the use of cells and tissues from a wide range of sources cultured in a three-dimensional (3D) setting resulting in more complex biological models. The application of these 3D models has been on the increase in areas such as medical research, precision medicine, disease modeling and drug discovery efforts. These models tend to be representative of the native microenvironments found in organisms and are thought to provide a more accurate assay model in some instances. 3D models are often compared to the widely used two-dimensional (2D) models, those consisting of a monolayer of cells, which have been in use for decades. The use of 2D assays has provided meaningful data but limitations became apparent and more recently the development of complex organoids, tissues or tumoroids comprised of one or more cell types, often based on stem cells or patient derived samples, have become the focus of many studies. The ability to perform a variety of assay types on a single instrument with a small footprint can be advantageous given the limited bench space found in many laboratories. The Agilent BioTek Cytation C10 confocal imaging reader performs microplate reading, widefield imaging as well as confocal imaging on a single instrument, providing a means to gather the wide range of data needed for many of today’s complex studies. This application note describes the use of an automated imaging assay for accurate determination of cell number in spheroids for evaluation of drug dose response

Materials and methods

HCT116-H2B-GFP cells were provided by the Stumpff lab at the University of Vermont. The H2B-GFP construct provided a constitutively expressing nuclear marker for quantification by cell count for comparison to a nuclear stain during live cell analysis. The cells were seeded at a relatively low density of 500 cells/well in a 96-well, round bottom ULA imaging plate, part number 4520, from Corning (Corning, NY) and allowed to grow for 3 to 4 days, depending on conditions. This resulted in spheroids of roughly 100 µm in size. The potent, nonselective inhibitor of protein kinases, staurosporin, was added as a known inducer of apoptosis. Propidium iodide (PI) was added simultaneously to monitor the events kinetically for determination of optimum analysis timing. It was determined that 12 hours post-addition was sufficient to show significant apoptotic activity for analysis


Figure 1. Spheroid assay workflow incorporating automated image acquisition and analysis conducted with the Agilent BioTek Cytation C10 confocal imaging reader and Agilent BioTek Gen5 microplate reader and imager software

Imaging procedure and analysis

The sizing of the spheroids was deliberate allowing the entire object to be captured, in a single field of view when using a 20x objective, to minimize the size of the data set. Additionally, a beacon was used to account for any variability in x-y positioning of the spheroid within the wells to keep to a single field of view. The 60 µm pinhole spinning disk was found to provide the best compromise between acquisition time and signal intensity for confocal imaging. Widefield images were captured in both GFP and TRITC channels for comparison to confocal images. The focus method used was “Scan”, followed by “Autofocus”. This focus method identifies the approximate center of the spheroid along the z-axis and allows each spheroid to be captured as a z-stack using 11 steps at 10 µm regardless of the z position in the microplate well. The images were subject to several processing steps including a z-projection for comparison of a single z-plane image at the approximate center of the spheroid and the entire z-stack for both imaging modes


Figure 2. The spheroid was captured as a z-stack using 11 steps at 10 µm intervals. Analysis was performed on either a z-projection of all the images or a single image representing a plane through the approximate center of the spheroid.

Automated cell counting was performed using confocal and widefield imaging modes, with analysis conducted on both single plane and z-stack projected images


Figure 3. Representative examples of automated image acquisition and cell count analysis of spheroids by confocal and widefield fluorescent microscopy using z-projected images. Cell masking represented by yellow outlines where GFP cell count indicates live cells and PI indicates dead cells


Figure 4. EC50 determinations for each imaging mode, single versus z-projected images


Spheroids are increasingly used to represent the more complex microenvironments indicative of biological systems. The ability to automate image capture using multiple imaging modes and perform automated image analysis can significantly increase throughput and allow for comparative analysis. Visual inspection of results confirmed confocal optics enable more accurate segmentation of labeled cells within spheroid samples compared to widefield optics. Cell count determination using a single z-plane image was found to provide comparable results to methods relying on z-stack image acquisition and processing. This study provides an example of the flexibility of the Agilent BioTek Cytation C10 confocal imaging reader and Agilent BioTek Gen5 microplate reader and imager software to capture and analyze multiple parallel data sets, using a single, compact instrument, to help simplify assay development and optimization

바이오테크랩 xCELLigence Cell Impedance

Measure Viral Cytopathic Effects (CPE) in Real Time Real-Time Virology Assays

Developing novel methods to screen antivirals is important for creating broad-spectrum antiviral drugs and vaccines. The accurate measurement of viral activity plays an essential role for viral vaccine development and infectious disease studies. When infected with a virus, host cells often display changes that are collectively referred to as a cytopathic effect (CPE). These effects include cell shrinkage or enlargement, deterioration/lysis, cell fusion, and the formation of inclusion bodies. Not all viruses cause a CPE in their host cell, but if they do, it can be a useful tool for various research applications from virus titer determination to the detection and quantification of neutralizing antibodies.

Explore Agilent xCELLigence Real-Time Cell Analysis technologies to learn about a viral cytopathic effect (CPE) assay that provides:

  • Rapid and highly sensitive kinetic data for the entire virus life cycle

  • A simple and high-throughput workflow, without the use of agar or labels

  • Reduced workload and manual handling of infectious samples through automation

  • More reproducible data by eliminating the subjectivity of visual interpretation

Fully Investigate Antiviral Drug Effects for the Entire Viral Life Cycle

Obtain highly sensitive and quantitative measurements of viral cytopathic effects, inside your incubator, with xCELLigence RTCA eSight. eSight provides multiplexed live cell imaging and impedance readouts that provide deeper insights into antiviral drug mechanism of action and capture cellular details spanning activation of host cell antiviral pathways, apoptosis, and barrier function disruption. Ensure crucial data and time points are never missed as you fully investigate drug impact over the entirety of the viral life cycle.

Multiplex Impedance-Based Data with Live Cell Imaging for Multiple Perspectives

바이오테크랩 xCELLigence Cell Impedance

While the standard xCELLigence RTCA instruments (SP, MP, HT models) provide comprehensive, non-invasive and label-free analysis in 96- or 384-well formats, the xCELLigence RTCA eSight adds live cell imaging for increased confidence in your cell analysis and conclusions. In one instrument, capture five different perspectives (impedance, brightfield, red, green, and blue fluorescence imaging) on the exact same population of cells to reveal greater insight into viral cytopathic effects.

Screen and Characterize Virus-Neutralizing Antibodies

바이오테크랩 xCELLigence Cell Impedance

An antibody’s ability to bind a purified antigen does not always correlate with its ability to “neutralize” the virus and block infection. In this example, xCELLigence RTCA impedance technology shows how a soluble CAR is unable to neutralize a virus when using 40k cells/well and the high virus concentration of MOI = 222. In contrast, when using 40k cells/well with the low virus concentration of MOI = 0.4, the soluble CAR completely blocks the ability of adenovirus-GFP to kill the host cells.

Real-Time Virology Assays

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