Sponsored Content by DeNovix Inc.Reviewed by Olivia FrostSep 13 2024
This article is based on a poster originally authored by DeNovix.
What is the best way to assess viability? Trypan Blue has long been the preferred method for selectively staining dead cells and tissues. However, for particular sample types, fluorescence assays such as Acridine Orange (AO) and Propidium Iodide (PI) can yield more precise results.
Trypan Blue
Trypan Blue is effective for determining the viability of cell samples with minimal debris. The dye cannot enter cells unless the membrane is damaged, meaning it only stains dead cells blue.
Live cells
Trypan Blue cannot pass through intact cell membranes; as a result, live cells remain unstained.
Image Credit: DeNovix Inc.
Dead cells
Trypan Blue stains cells with damaged membranes blue.
Image Credit: DeNovix Inc.
AO/PI fluorescence
AO is a nucleic acid-binding fluorophore that can pass through cell membranes, staining the nuclei of all cells in a sample.
PI is a nucleic acid-binding dye that cannot penetrate live cell membranes, but it is suitable for staining dead, nucleated cells.
Live cells
In live cells, AO permeates the cell membrane, staining the nuclei and causing the cell to fluoresce green.
Image Credit: DeNovix Inc.
Dead cells
AO and PI stain dead nucleated cells, causing them to fluoresce red due to FRET.
Image Credit: DeNovix Inc.
Recommended sample types
Use with Trypan Blue or AO/PI
- Samples with minimal debris
- Tissue culture samples
AO/PI Recommended
- Primary cells
- Whole blood samples
- Samples with significant debris
- Nuclei counting
- Yeast and small cells
Image Credit: DeNovix Inc.
Sample preparation
Trypan Blue
- Optional: filter the Trypan Blue solution through a 0.2 µm filter to eliminate any aggregates or crystals that may form.
- Add Trypan Blue to the cell suspension in a 1:1 ratio and mix.
- Thoroughly mix the sample just before loading.
AO/PI fluorescence
- Equilibrate all solutions to room temperature.
- Add AO/PI to cell suspension in a 1:1 ratio and mix.
- Thoroughly mix the sample just before loading.
Recommended instruments and apps
The CellDrop™ Automated Cell Counter measures cell viability using brightfield and fluorescence technologies. The CellDrop BF is equipped for brightfield counting applications, while the CellDrop FLi (brightfield and fluorescence) offers applications for both brightfield and fluorescence-based counting.
Trypan Blue CellDrop count
In brightfield, live cells appear with a bright center and a black membrane, while Trypan Blue stains dead cells, giving them a dark appearance. Live cells must be distinguished from debris either manually or through counting algorithms.
Image Credit: DeNovix Inc.
AO/PI CellDrop count
Cells labeled with AO/PI either fluoresce red (dead) or green (live). Debris and non-nucleated cells are not stained, which eliminates subjectivity from the analysis.
Image Credit: DeNovix Inc.
Data comparison
CHO cells were counted using Trypan Blue and AO/PI. Both methods can accurately count CHO cells, and cell counts for each dilution are comparable.
Image Credit: DeNovix Inc.
PBMCs were counted using Trypan Blue and AO/PI. AO/PI provides an accurate count of PBMCs, whereas Trypan Blue tends to overestimate cell numbers by incorrectly counting cellular debris and non-nucleated red blood cells.
Image Credit: DeNovix Inc.
Summary
Brightfield measurements with Trypan Blue (or similar colorimetric dyes, such as Erythrosin B) effectively report cell counts and viability in cultured cell lines. However, the dye has limitations in distinguishing cells from debris.Dual fluorescence measurements using AO/PI allow for precise identification of live and dead cells, even in the presence of large amounts of non-nucleated cells and debris. This eliminates the subjectivity of colorimetric dyes and enhances the overall accuracy of the results.
CellDrop
The CellDrop Automated Cell Counter utilizes DirectPipette™ technology, eliminating the need for disposable plastic slides. This equipment includes dual fluorescence and brightfield optics, a variable height sample chamber, and simple analysis software.
Download this infographic from DeNovix to learn about viability methods, sample compatibility, and how automated cell counters like the DeNovix CellDrop™ deliver accurate and consistent measurements.
Download the full poster to learn more
About DeNovix, Inc.
WELCOME TO DENOVIX
Award-Winning products for Life Science
Our multi-award winning products include the Reviewers’ Choice Life Science Product of the Year and Platinum Seal awarded- DS-11 Series Spectrophotometer / Fluorometer and CellDrop™ Automated Cell Counter. CellDrop is the first instrument of its kind to Count Cells Without Slides. These powerful instruments integrate patented DeNovix technology with easy-to-use software designed by life scientists for life scientists.
Researchers tell us they love the industry leading performance, smart-phone-like operation, and the flexible connectivity of the instruments. When support is needed, the DeNovix team is here to help. DeNovix received the prestigious Life Sciences Customer Service of the Year based on independent reviews posted by scientists worldwide!
CellDrop: Sustainable laboratory product of the year
The CellDrop Automated Cell Counter has been awarded Sustainable Laboratory Product of the Year in the SelectScience® Scientists’ Choice Awards®!
CellDrop’s patented DirectPipette™ technology distinguishes it as the only cell counter to eliminate the need for cell counting slides. This innovation saves millions of single-use plastic slides from use and disposal each year.
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