Cell Counting

Learn how to count cells using a variety of methods, techniques, and technologies

Cell Counting Methods and Techniques

The ability to accurately quantitate cell number in multi-well microplates enables a multitude of biological applications that study cell health or proliferation. These applications may make use of endpoint assays for imaging fluorescently-stained nuclei, or may demand robust transmitted light imaging of unstained live or fixed cells. In both cases, the enumeration of the cells through software segmentation should be fast and reliable.

Here, we discuss the various methods and techniques used to assess proliferation, cytotoxicity, and confluence using cell counting, which can be quickly accomplished with either brightfield or fluorescent imaging using an automated imaging system and analysis software.

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.

Automated cell imaging

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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.

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Stainfree cell detection technology

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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Resources of Cell Counting

Automated cell counters, like the SpectraMax® i3x Multi-Mode Microplate Reader with SpectraMax MiniMax cytometer or the ImageXpress® Pico Automated Cell Imaging System, can assess proliferation, cytotoxicity, or confluency with either brightfield or fluorescent imaging.

SpectraMax i3x Multi-Mode Microplate Reader with SpectraMax MiniMax cytometer enables quick imaging and analysis of cells and gives you a front-row view of phenotypic changes that accompany cytotoxicity, cell proliferation, and protein expression. Our StainFree Cell counting Technology now includes fluorescent green and red detection channels and additional brightfield analysis features.

ImageXpress Pico Automated Cell Imaging System is an automated cell counter using transmitted light or fluorescent imaging to capture data-rich outputs such as nuclear size, total and average cell area, and intensity. In addition to the standard cell counting methods for detecting fluorescent nuclei or cell bodies, the software includes three additional protocols for counting cells in transmitted light based on cell size and shape.