Cell Counting Methods and Techniques
Cell counting using automated cell imaging
Cell counting is fundamental and critical to numerous biological experiments. Assays such as drug compound toxicity, cell proliferation, and inhibition of cell division have a need to assess the number or density of cells in a well. Automated imaging can greatly speed up the cell counting process while reducing manual labor and human errors.
The example workflow below shows that a user can simply place the sample into the ImageXpress® Pico Automated Cell Imaging System, and after a simple setup in the software, the system can count cells automatically, allowing users to have walkaway time. Cells can be counted using a variety of methods such as label-free cell counting under transmitted light, or detection of nuclear dye with fluorescent imaging.

StainFree Cell Detection Technology: A label-free method for analyzing cell counts and cell confluence
Imaging cell-based assays typically requires the use of fluorescent probes that can be toxic to living cells or may only function in fixed cells. A label-free method for analyzing cell counts and cell confluence enables you to quantitatively monitor cell proliferation and health without time-consuming workflows that may disrupt cell viability.
The SpectraMax® i3 Multi-Mode Microplate Reader with the SpectraMax® MiniMax™ 300 Imaging Cytometer uses unique, patent pending, StainFree™ Cell Detection Technology that allows you to perform cell proliferation, cytotoxicity, and other assays without nuclear stains like DAPI, which intercalates with DNA, or live cell dyes that are actually toxic to cells in the long term.

Workflow for cell analysis with StainFree technology vs. Live Red Dye vs. DAPI. The StainFree workflow saves about 70 minutes compared to fixation and staining with DAPI. Moreover, cells analyzed using StainFree technology remain fully viable and can be used in additional assays.
Counting cells with StainFree technology
The StainFree cell detection algorithm eliminates cell staining for cell counting and confluence measurements using proprietary transmitted light (TL) analysis technology. Here, we describe the analysis of a variety of commonly used cell types using our label-free method.
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Access cell viability
Cell viability can be assessed by examining parameters such as cell membrane integrity or the activity of cellular enzymes. On a microplate readers, these parameters can be detected using fluorescent reagents. For example, a red cell-impermeant, DNA-binding dye only stains dead or dying cells whose membranes are compromised, while a green live-cell dye only fluoresces when metabolic enzymes are active.
Alternatives to DAPI staining
DAPI (4’, 6-diamidino-2-phenylindole) is a fluorescent dye often used to stain nuclear DNA. It is employed in imaging experiments such as fluorescent microscopy, chromosome spreads, FACS, and cell-based assays. However, excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI fluorescence in the FITC/GFP channel of an imaging system, causing errors in interpretation of results. DAPI also requires cells to be fixed for maximal staining. Here we show some alternatives to DAPI staining, including StainFree technology, which requires no staining at all and can be used with live cells.
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Calculate transfection efficiency
Measuring transfection efficiency provides researchers with an early indication of their gene editing yield before using the modified cells for targeted assays. High quality images can be acquired with both the transmitted light and with a fluorescence channel indicating transfection of the cell. Image analysis software identifies and counts every cell plus scores them as GFP-positive or not. Afterwards, a transfection efficiency may be calculated from the ratio.
Cell cycle analysis
Transition of a cell through the cell cycle can be monitored using fluorescently-labeled cell cycle proteins that are cyclically expressed and degraded. FUCCI technology is based on the overexpression the cell cycle-dependent proteins geminin and Cdt1, respectively fused to a green fluorophore and a red fluorophore. Cdt1 levels peak in G1 phase, so cells in G1 appear green; geminin levels rise in late S, G2 and M phase, so cells in these phases appear red.
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Cell migration
Cell migration, the movement of cells from one location to another, is a critical component of both normal and abnormal biological processes. The migration of unlabeled or fluorescently labeled cells from one area of a microplate well to another can be monitored using a cellular imaging system. Automated analysis software calculates the amount of cell migration in each well.
Count cells with fluorescent markers
Image and analyze compound effects based on cells staining positive for different fluorescent markers. The consistent and statistically relevant quantitative analysis allows for the testing of multiple compounds at differing concentrations. Read the application note to learn more about the difference between cell counting using fluorescent or transmitted light imaging.
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Counting cells with transmitted light
Transmitted light segmentation (analysis) algorithms increase the accuracy of counting diverse cell types and are typically used to image unstained live or fixed cells. In addition to the standard cell counting methods for detecting fluorescent nuclei or cell bodies. Watch the video to learn how you can set up parameters to run transmitted light cell scoring.
- Transmitted light image analysis for cell count and assessment of cytotoxicity effects
- Improved cell counting method using transmitted light on the SpectraMax MiniMax cytometer
- Transmitted light cell scoring on the ImageXpress Pico
- Counting cells with the Transmitted Light Analysis Module in MetaXpress
Detect cytotoxicity in cells
Cytotoxicity is often measured in response to an experimental treatment or potential drug. Having a way to easily screen cytotoxicity in treated cells is critical to identifying new therapeutic treatments or understanding cellular signaling pathways that affect cell health. Common indicators of cytotoxicity include the ATP level in a cell population and integrity of cellular membranes, both of which can be measured using a variety of microplate-based assays.
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eBook: Count cells like a pro
Improve cell counts by adding cellular imaging assays in a small footprint and eliminating cell staining and confluence measurements with StainFree™ Cell Detection Technology.
Image cytometry
Image cytometry is a cell counting technique that combines low magnification microscopy with imaging and analysis tools. Image cytometry is useful for providing information on a broad range of cellular characteristics on a large population of cells in a single assay. Image cytometry can be used on unlabeled or fluorescently labeled cells and can be performed multiple times on a population to provide information about cellular dynamics over time.
Resources of Cell Counting
Application Note
Spectral Fusion™ Illumination technology for an extended dynamic range on the SpectraMax i3x Multi-Mode Microplate Reader
Spectral Fusion™ Illumination technology for an extended dynamic range on the SpectraMax i3x Multi-Mode Microplate Reader
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CloneSelect Imager FL fluorescence for rapid day zero monoclonality
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Cellular Imaging Insights
Cellular Imaging Insights
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Measuring cell viability and cytotoxicity with the EarlyTox Cell Integrity Kit
Measuring cell viability and cytotoxicity with the EarlyTox Cell Integrity Kit
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Improving acquisition and analysis of 3D cell model assays with water immersion objectives
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The workflow game-changer: combined technologies for efficiency
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What 3D imaging means for cancer drug discovery
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Application Note
Measure cell migration using a simple scratch assay with timelapse live-cell imaging
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Harnessing the latest imaging technology to advance angiogenesis research
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High-content screening of complex physiologically-relevant cell models
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Application Note
Count cells with or without fluorescent labels using automated imaging
Count cells with or without fluorescent labels using automated imaging
The ability to accurately quantitate cell number in multi-well microplates enables a multitude of biological applications that study cell health or proliferation.
Application Note
Measure cell migration using discontinuous time-lapse imaging of live cells
Measure cell migration using discontinuous time-lapse imaging of live cells
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Normalize HTRF cytokine assays to cell viability
Normalize HTRF cytokine assays to cell viability
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Counting cells with the Transmitted Light Analysis Module in MetaXpress
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Scientific Poster
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Improved cell counting method using transmitted light on the SpectraMax MiniMax cytometer
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Measuring cell health on the SpectraMax iD3 reader with cell viability assays
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Transmitted light image analysis for cell count and assessment of cytotoxicity effects
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Application Note
Evaluating cell cycle inhibitors using a live cell assay
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Validate CRISPR-Edited Cells using Imaging and Western Blot Detection on a Microplate Reader
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Evaluate cell migration with FluoroBlok inserts on the SpectraMax MiniMax cytometer
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eBook
Cellular Imaging Made Easy
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eBook
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Count Cells Like a Pro
Improve cell counts by adding cellular imaging assays in a small footprint and eliminating cell staining and confluence measurements with StainFree™ Cell Detection Technology.
Application Note
Cytotoxicity in Cells: Easy Determination Using Lonza ViaLight Plus and ToxiLight BioAssays on the SpectraMax L Microplate Luminometer
Cytotoxicity in Cells: Easy Determination Using Lonza ViaLight Plus and ToxiLight BioAssays on the SpectraMax L Microplate Luminometer
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Application Note
Monitoring cytotoxicity on the SpectraMax i3 platform with MiniMax cytometer
Monitoring cytotoxicity on the SpectraMax i3 platform with MiniMax cytometer
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Application Note
Assessing mitochondrial toxicity on the SpectraMax i3 Multi-Mode Detection Platform with SpectraMax MiniMax 300 Imaging Cytometer
Assessing mitochondrial toxicity on the SpectraMax i3 Multi-Mode Detection Platform with SpectraMax MiniMax 300 Imaging Cytometer
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Wells to Westerns: Investigating the cellular heat shock response
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Application Note
Alternatives to DAPI staining: imaging and counting live cells
Alternatives to DAPI staining: imaging and counting live cells
DAPI (4’, 6-diamidino-2-phenylindole) is a fluorescent dye often used to stain nuclear DNA. It is employed in imaging experiments such as fluorescent microscopy, chromosome spreads, FACS,…
Application Note
StainFree technology for cellular image analysis without fluorescent dyes
StainFree technology for cellular image analysis without fluorescent dyes
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Application Note
Optimizing GFP transfection with the SpectraMax i3 Multi-Mode Detection Platform
Optimizing GFP transfection with the SpectraMax i3 Multi-Mode Detection Platform
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Application Note
Multiplexed high-content hepatotoxicity assays using iPSC-derived hepatocytes
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Videos & Webinars

Transmitted light cell scoring on the ImageXpress Pico