Fluorescence Cell Counting

Fluorescence-based cell counting has emerged as a highly accurate and efficient method for quantifying cell populations. This article explores the principles and advantages of fluorescence cell counting before looking at some of the technologies available to researchers.

Numerous applications depend on cell counting

Cell counting is essential for applications spanning basic research through to clinical practice. Its uses encompass routine cell culture and passaging; functional assays such as cytotoxicity, viral infectivity, and gene vector transduction efficiency; tracking of cell proliferation, particularly in cancer research; and air and water quality monitoring.

“Cell counting and viability measurements also serve as critical quality attributes for cell and gene therapy (CGT) product manufacturing processes,” reports Bo Lin, Ph.D., Leader for Strategic Collaborations and Opportunities at Revvity. “For many new modality medicines, such as CGT, effectiveness is closely linked to cell quality and quantity.”

Besides cell counts, nuclei counts are becoming an important application for those performing snRNA-seq, ATAC-seq, and other related techniques “Due to the nature of these methods, researchers need accurate nuclei counts, live cell quantitation to confirm successful nuclei harvesting, and cluster analysis to verify how reliably the method yields single nuclei,” says Andrew Jones, Market Development Manager at DeNovix.

Cell counting with chromogenic dyes

Staining cells with Trypan Blue and loading them into a hemocytometer for manual inspection under a microscope is an established method for cell counting. Because Trypan Blue only enters cells with damaged membranes, it provides a quick and easy means of excluding non-viable cells from the total cell count. Alternatively, researchers can use Erythrosin B for chromogenic cell counting.

“Compared to Trypan Blue, Erythrosin B has lower toxicity, better preserving cells for further experiments,” reports Seungyo Jeong, Business Development at NanoEntek. “Its reduced hazard profile and minimal environmental impact make Erythrosin B a dependable alternative for cell counting.”

In many labs, chromogenic cell counting methods have been adapted for use with automatic cell counting systems. “Automatic systems provide consistent and accurate results by standardizing the cell counting process and reducing human error,” says Lin. Additionally, researchers are choosing fluorescence cell counting for the advantages it can provide.

Principles of fluorescence cell counting

Fluorescence cell counting typically involves the use of nuclei acid-binding fluorescent dyes. “Generally, the most effective cell counters employ a dual-fluorescence approach using dyes such as Acridine Orange (AO) and Propidium Iodide (PI),” explains Jones. “These respectively fluoresce green and red upon binding to DNA. However, while AO will enter all cells, PI will only enter cells when membrane integrity is impaired.” As a result of Förster resonance energy transfer (FRET), whereby PI absorbs AO emission, dead cells selectively yield only red fluorescence.

Another strategy combines nucleic acid staining with the detection of metabolic activity. For example, ethidium homodimer-1, a dye that enters cells with compromised membranes and produces red fluorescence upon binding to DNA, is often paired with Calcein AM, a cell-permeant reagent that fluoresces green following its cleavage by intracellular esterases. In both scenarios, cell viability is calculated by dividing the number of green fluorescent cells by the total number of cells and multiplying by 100.

Advantages of fluorescence cell counting

According to Lin, a limitation of Trypan Blue and other non-fluorescence-based methods is that they can't reliably differentiate intact nucleated cells from non-nucleated red blood cells or aggregated platelets. “With fluorescence, researchers can confidently count various types of samples, including tumor-digested cells, isolated nuclei, and fresh or cryopreserved PBMC samples,” she says.

“Another important advantage of fluorescence cell counting is that it provides higher contrast from the background, making the cells easier to identify and count,” says Kristi Hamilton, Ph.D., Scientist at Thermo Fisher Scientific. “Fluorescence cell counting also reduces the likelihood of misidentifying debris as cells, ensuring more accurate results, and allows for differentiating and counting various cell types using different fluorescence markers, increasing flexibility for scientific research.”

“A further benefit of fluorescence-based cell counting methods is that they are readily scalable, making them ideal for high-throughput and clinical applications,” notes Jeong. “In recognition of the advantages just described, the Korean Ministry of Food and Drug Safety recently updated its cell therapy product quality control guidelines to officially include fluorescence-based cell counting as an approved method.”

PBMCs counted with Trypan Blue and AO/PI. PBMCs are accurately counted with AO/PI. Cell counts are overestimated when counting with Trypan Blue due to the incorrect counting of cellular debris and non-nucleated red blood cells. Image provided by DeNovix.

Fluorescence cell counting solutions

Technologies for fluorescence cell counting include Thermo Fisher Scientific’s Countess™ 3 FL Automated Cell Counter,  which features unique AI-driven counting algorithms and requires only 10 µL of sample to deliver results in less than 30 seconds. “The Countess 3FL offers automated auto-focusing and auto-lighting, while also allowing custom programming for user-specified exposure times,” says Hamilton. “In addition, users can gate out unwanted cells based on size, fluorescence, or shape, ensuring precise and accurate results.”

The CellDrop™ Automated Cell Counter from DeNovix  is an image-based instrument that is unique in removing the need for disposable plastic slides or cartridges from the cell counting process. “This decreases laboratory costs, reduces the sample processing time, and has a significant environmental benefit by reducing the reliance on disposable plastic,” says Jones. Notably, the CellDrop is the first cell counter to receive the ACT environmental impact label.

NanoEntek’s range of cell counters includes the EVE HT FL, a high-throughput dual fluorescence instrument that enables fast, accurate, and automated cell counting through advanced image analysis technology. “The EVE HT FL measures up to 48 samples within 3 minutes, using just 20 µL per sample,” says Jeong. “It is based on a cell nucleus fluorescent staining method and delivers precise results, unaffected by cell type, size, or contaminants.”

Revvity’s cell counting solutions combine advanced imaging technologies with proprietary pattern-recognition software, quickly identifying and counting individual cells, and automatically calculating cell concentration, diameter, and viability. With the 21CFR Part 11 module, the fluorescent counters operate in GLP/GMP environments. Products include the Cellometer Ascend for small precious samples, the Cellaca MX for high throughput, and the Celigo image cytometer for directly analyzing cells in plates for 2D, 3D, or organoid experiments.

Key factors to consider when selecting a fluorescence cell counter

• What types of samples do you want to count?
• What level of throughput do you need?
• Do your cell types need specific algorithms to be counted successfully?
• Do you wish to avoid using disposable plastics?
• Would you like to efficiently upscale using automation?
• What is the instrument footprint, cost, and capacity for future upgrades?