How to improve cell counting?

Trypan Blue is widely used in cell culture laboratories for selectively staining dead cells and tissues. However, the utilization of this dye may pose challenges when accurately evaluating cell counts and viability in certain sample types.

Without implementing additional time-consuming and costly purification steps, primary cell samples are likely to contain debris and non-nucleated cells, which will be difficult to count with trypan blue.

The DeNovix Acridine Orange (AO) / Propidium Iodide (PI) fluorescence viability assay is a user-friendly and more accurate alternative for these sample types.

To discover how to improve cell counting accuracy, download this free eBook.

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Trypan Blue cell counts

Trypan Blue effectively assesses the viability of cell samples with minimal debris. The dye is prevented from entering cells unless the membrane is damaged. In this assay, dead cells are stained a distinctive blue color, while live cells remain transparent.

The Trypan Blue app on the DeNovix CellDrop™ Automated Cell Counter counts live and dead cells, determines cell concentration, and reports viability. The software detects live cells as objects featuring a bright white center enclosed by a sharp dark ring. Stained cells are detected as objects that appear dark in contrast to the background.

Trypan Blue is most effective for samples without debris, such as tissue culture cells trypsinized from a flask or plate. In samples with debris, distinguishing between live cells and debris becomes challenging and subjective. The viability and counts of these samples are frequently overestimated when debris is erroneously included in the count and viability calculation.

AO/PI cell counts

AO is a cell membrane-permeable nucleic acid-binding fluorophore. This dye stains the nuclei of all cells in a sample. PI is a nucleic acid-binding dye that cannot permeate live cells; however, it is well-suited for staining dead, nucleated cells.

The DeNovix AO/PI Viability Assay combines both dyes and is optimized for precise counting and viability evaluation of nucleated cells. The CellDrop FL Automated Cell Counter features an AO/PI app, which utilizes images from the brightfield, green fluorescence, and red fluorescence channels.

Live, nucleated cells exhibit green fluorescence due to AO. Dead, nucleated cells are stained with PI and AO, resulting in red fluorescence due to Förster resonance energy transfer (FRET) quenching of the AO dye by the PI dye. Non-nucleated cells, along with cellular debris, remain unstained.

The AO/PI assay proves highly effective for analyzing nucleated cells in samples containing debris. For example, peripheral blood mononuclear cells (PBMCs) are difficult to quantify with brightfield techniques like the Trypan Blue assay. This is because of the high background of non-nucleated red blood cells, which the dye fails to differentiate.

CHO cells counted with Trypan Blue. Tissue culture cells accurately counted with Trypan Blue

Figure 1. CHO cells counted with Trypan Blue. Tissue culture cells accurately counted with Trypan Blue. Image Credit: DeNovix Inc.

PBMCs counted with AO/PI. Brightfield / Green / Red image overlay for PBMCs counted with AO/PI accurately determine the viability of samples with debris.

Figure 2. PBMCs counted with AO/PI. Brightfield / Green / Red image overlays for PBMCs counted with AO/PI accurately determine the viability of samples with debris. Image Credit: DeNovix Inc.

Assessing these samples with AO/PI is easier and more accurate. Within these complex samples, nucleated cells are easily distinguishable by green fluorescence (indicating live cells) or red fluorescence (indicating dead cells) against a background of non-fluorescing cells (shown in Figure 2).

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About DeNovix, Inc.


DeNovix Inc. is an instrumentation company that designs, manufactures and sells laboratory equipment to meet the demands of today’s evolving life science technologies. Our focus is on providing innovative products and outstanding customer support. DeNovix is equipped with the financial, commercial and technical resources to deliver breakthrough products for your research success.

Our multi-award winning products include the DS-11 Series Spectrophotometer / Fluorometer and CellDrop™ Automated Cell Counter. Each instrument is a fully networkable, stand-alone system with an HD touchscreen and custom EasyApps™ software designed by life scientists for life scientists. Labs love the smart-phone-like operation, impressive performance, and the flexible connectivity of the instrument. 

Learn more about DeNovix instruments and how they can optimize your research workflows.

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Last updated: Feb 4, 2024 at 9:28 PM


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