In cell culture laboratories, Trypan Blue dye is commonly used for selective staining of dead cells and tissues. However, using this dye can pose challenges in accurately assessing cell count and viability in certain types of samples.
Primary cell samples that have not undergone additional complex and time-consuming purification steps often contain contaminants and non-nuclear cells that are difficult to count using Trypan Blue.
The viability assessment method using fluorescence dyes Acridine Orange (AO) and Propidium Iodide (PI) offered by DeNovix provides a user-friendly and more accurate alternative for such samples.
To learn more about improving cell counting accuracy, download the free ebook.
Cell Counting with Trypan Blue
Trypan Blue effectively assesses the viability of cell samples by minimizing the presence of contaminants. The dye does not penetrate live cells unless the cell membrane is damaged. In this test, dead cells are stained a characteristic blue color, while live cells remain transparent.
The Trypan Blue application available on the DeNovix CellDrop™ automated cell counter allows for counting live and dead cells, determining cell concentration, and performing viability assessment. The software detects live cells as objects with a bright white center surrounded by sharply defined dark outlines. Stained cells are detected as objects that appear dark in contrast to the background.
Trypan Blue is most effective in clean samples, such as cultured tissue culture cells after detachment from flask or plate. In samples containing contaminants, it is difficult to differentiate live cells from contaminants, leading to overestimation of both cell count and viability.
Cell Counting with AO/PI
AO is a dye that binds to nucleic acids and is permeable to the cell membrane. This dye stains the nuclei of all cells in the sample. PI is a dye that also binds to nucleic acids but does not penetrate live cells. It is ideal for staining dead, nuclear cells.
The AO/PI viability assay developed by DeNovix combines both dyes and is optimized for precise counting and assessment of nuclear cell viability. The CellDrop FL automated cell counter is equipped with the AO/PI application, which utilizes images from brightfield, green fluorescence, and red fluorescence channels.
Live, nuclear cells exhibit green fluorescence from AO dye. Overlapping Förster resonance energy transfer (FRET) between AO and PI dyes causes dead, nuclear cells to be stained in red. Non-nuclear cells, as well as cellular contaminants, remain unstained.
The AO/PI assay works exceptionally well for analyzing nuclear cells in samples containing contaminants. For example, peripheral blood mononuclear cells (PBMCs), which are difficult to count using brightfield techniques like Trypan Blue, can be accurately counted using AO/PI. This is due to the high background of non-nuclear red cells that the dye cannot distinguish.
Counting these samples using AO/PI is simpler and more accurate. Within these complex samples, nuclear cells are easily distinguishable through either green fluorescence (indicating live cells) or red fluorescence (indicating dead cells) against non-fluorescent cells (as shown in Figure 2).
About DeNovix, Inc.
DeNovix Inc. is a company dedicated to manufacturing laboratory equipment that meets the requirements of today’s scientific technologies. Their goal is to provide innovative products and excellent customer service. DeNovix has the necessary financial, commercial, and technical resources to deliver groundbreaking products that contribute to the success of your research.
DeNovix’s award-winning products include the DS-11 Series spectrophotometers/fluorometers and the CellDrop™ automated cell counter. Each device is a fully networked, stand-alone system with a HD touch screen and EasyApps™ software specifically designed by scientists for scientists. Laboratories appreciate the instrument’s intuitive operation, impressive performance, and wide range of connectivity options.
To learn more about DeNovix instruments and how they can optimize your research processes, visit the DeNovix website.
FAQ Section based on key topics and information presented in the article:
1. What is Trypan Blue and how is it used in cell culture?
Trypan Blue is a dye commonly used for selective staining of dead cells and tissues in cell culture laboratories. It is used to assess cell count and viability in samples.
2. How does Trypan Blue work in cell viability assessment?
Trypan Blue does not penetrate live cells unless their cell membrane is damaged. Dead cells are stained blue, while live cells remain transparent.
3. What are the challenges associated with using Trypan Blue in cell counting?
Primary cell samples contain contaminants and non-nuclear cells, making it difficult to accurately count using Trypan Blue.
4. What is the fluorescence method of cell counting using AO and PI dyes?
The viability assessment method using fluorescence dyes Acridine Orange (AO) and Propidium Iodide (PI) is a more accurate alternative for samples containing contaminants. AO stains the nuclei of all cells, and PI stains dead, nuclear cells.
5. How does the AO/PI test work in cell counting?
The AO/PI test utilizes green fluorescence from the AO dye to label live cells and red fluorescence from the PI dye to label dead cells. This allows for precise cell counting and viability assessment.
6. Why is the AO/PI test more effective than Trypan Blue in samples with contaminants?
Trypan Blue is difficult to differentiate live cells from contaminants in samples with contaminants. However, the AO/PI test is more accurate because nuclear cells are easily distinguishable through green and red fluorescence.
7. What does DeNovix, Inc. specialize in?
DeNovix Inc. specializes in manufacturing laboratory equipment that meets the requirements of today’s scientific technologies. Their products, such as the DS-11 Series spectrophotometers/fluorometers and the CellDrop™ automated cell counter, are award-winning and offer innovative solutions for laboratories.
8. What are the benefits of using DeNovix instruments?
DeNovix instruments, such as the CellDrop™ automated cell counter, are equipped with advanced applications that allow for accurate cell counting and viability assessment. They also feature intuitive interfaces, high-quality screens, and a wide range of connectivity options.
Suggested Related Links (main domain):
– DeNovix website
Definitions of key terms and jargon:
– Trypan Blue: A dye used for selective staining of dead cells and tissues in cell culture.
– Acridine Orange (AO): A fluorescent dye that stains the nuclei of all cells in a sample.
– Propidium Iodide (PI): A fluorescent dye that stains dead, nuclear cells.
– FRET (Forster Resonance Energy Transfer): Overlapping energy transfer between AO and PI dyes that leads to staining of dead, nuclear cells in red.
– DeNovix CellDrop™: DeNovix’s automated cell counter.
Suggested Related Links:
– CellDrop – automated cell counter
– DS-11 Series – spectrophotometers/fluorometers
The source of the article is from the blog kunsthuisoaleer.nl