Why working with trypan blue is not a good idea
A review of cell count accuracy and viability using trypan blue
Does trypan blue give an accurate measurement of cell viability?
For decades, cells counted under a brightfield microscope have been stained by trypan blue to estimate cell viability. This method relies on the nuclear-exclusion principle: Membrane-impermeable trypan blue is excluded from viable cells but enters permeable dead cells.
Unfortunately, after only a short exposure time of five minutes, trypan blue starts to become toxic to cells1, 2, 3. Even viable cells are eventually stained with trypan blue, as when the dye permeates their cell membrane, they die over time (normally between 5 to 30 minutes after exposure).
The toxicity of trypan blue impacts the accuracy of measurements in two ways: (i) the viability analysis changes over time, and (ii) the viability of a cell population is underestimated2.
Alternative, more accurate methods exist
Brightfield microscopy is used to validate and quantify cell cultures. It gives ambiguous staining patterns and relies on a series of parameters, including roundness and uniformity, to discriminate cells from non-cellular particles. Trypan blue stains dead cells in shades ranging from light blue to black, depending on the overall viability of the cell culture. When using a brightfield microscope, cells that are very lightly stained with trypan blue can be hard to differentiate from unstained cells, and thus hard to identify. Also, individual lab members will each have a somewhat subjective view of what is a dead versus a living cell. Therefore, using trypan blue to distinguish between live and dead cells introduces the risk of variation between users and instruments.
For these reasons, fluorometric assays are considered a better choice for the evaluation of cell viability than colorimetric ones, such as trypan blue4. Because cells have a relatively fixed amount of DNA/RNA, the DNA-interacting fluorophores acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) will produce a uniform staining pattern for the quantification of cells, compared to the unspecified staining pattern when using brightfield-based analysis. AO is membrane-permeable and stains the total cell population, whereas DAPI only stains the dead cell population. Therefore, fluorometric determination of cell viability using AO and DAPI provides superior accuracy to identify live and dead cells with reduced noise and variation.
Ensuring a safe working environment is crucial for businesses. According to the European Chemical Agency (ECHA), trypan blue may cause cancer, genetic defects, and damage fertility, or cause harm to an unborn child5. Consequently, special precautions should be taken to avoid exposing operators to trypan blue whilst working with and disposing of the dye.
Another important aspect to consider when handling trypan blue is that it is classified as a Group 2B carcinogen (ECHA5). This makes working with trypan blue potentially hazardous for operators in the lab since exposure should be avoided when possible.
There are safe cell counting alternatives that provide increased precision and improved accuracy.
The Via2-Cassette™ is a unique microfluidic device that provides unparalleled accuracy and precision of a cell count and eliminates a user’s exposure to chemicals. The Via2-Cassette™ includes a built-in pipette that prevents errors during pipetting and staining. The fluorescent dyes AO and DAPI are immobilized inside the cassette’s channels, automatically staining the total and dead cell populations, respectively. During the entire cell counting process, AO and DAPI are kept inside the microfluidic device, significantly reducing risk of exposure. The Via2-Cassette™ is disposed of after every use, helping to maintain a safe working environment.
The Via2-Cassette™ is used with the NucleoCounter® NC-202™, an automated fluorescent cell counter that provides an analysis of the total and live cells, the dead cell concentration, viability, and the DebrisIndex™ of a sample, all in approximately 30 seconds. The instruments are constructed using high-quality components that remain stable over time, which together with extensive calibration procedures performed at the time of build, results in low variation between instruments, users and sites. This technology delivers an optimal level of precision to your cell counting.
The NucleoCounter® NC-202™ with the Via2-Cassette™, combines industry-leading accuracy and precision that delivers consistent results while keeping users safe from harm.
- JR Tennant: Evaluation of the Trypan Blue Technique for Determination of Cell Viability. Transplantation, 1964. 2: p. 685-94.
- KT Tsaousis, N Kopsachilis, IT Tsinopoulos, SA Dimitrakos, FE Kruse, U Welge-Luessen: Time-dependent morphological alterations and viability of cultured human trabecular cells after exposure to Trypan blue. Clin Exp Ophthalmol, 2013. 41(5):484-90.
- AKH Kwok, C-K Yeung, TYY Lai, K-P Chan, CP Pang: Effects of trypan blue on cell viability and gene expression in human retinal pigment epithelial cells. Br J Ophthalmol. 2004;88(12):1590-4.
- SA Altman, L Randers, G Rao: Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnol Prog. 1993;9(6):671-4.
- European Chemical Agency (ECHA): Substance Infocard: Tetrasodium 3,3′-[(3,3′-dimethyl[1,1′-biphenyl]-4,4′-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonate]