Trypan Blue is an azo dye used in cell viability assays to distinguish live and dead cells with its membrane exclusion mechanism.

Trypan Blue leverages its ability to differentiate live and dead cells based on membrane integrity: viable cells with intact membranes exclude the dye and remain unstained, whereas non-viable cells with compromised membranes absorb the dye and appear blue under the microscope. This straightforward exclusion assay involves mixing a cell suspension with Trypan Blue, incubating briefly, and counting stained (non-viable) and unstained (viable) cells under a light microscope. This rapid and accurate method enables researchers to efficiently assess cell health across various biological and experimental settings.

Cancer Research—Trypan Blue continues to be integral in cancer cell studies, specifically in evaluating the effectiveness of anti-cancer compounds in killing tumor cells while preserving normal cell viability. (Del Grande et al., 2022)

3D Cell Culture—As 3D cell culture models become increasingly important for mimicking in vivo environments, Trypan Blue is being adapted to assess cell viability within these complex structures. (Smith et al., 2021)

Gene Editing Validation—The dye is also applied to verify cell viability after gene editing processes, such as CRISPR/Cas9, ensuring minimal off-target effects or cytotoxicity. (de Oliveira et al., 2020)

Cell Therapy Quality Control—Trypan Blue is increasingly being utilized for evaluating the viability of stem cells or engineered cells prepared for therapeutic use. This application ensures that cells modified for treatments, such as CAR-T cell therapy or stem cell-based regenerative medicine, maintain high viability and functional integrity. (Jones et al., 2024)

Simple and Cost-Effective—A reliable method that requires minimal preparation and is easy to implement in standard laboratory settings.

Rapid Assessment—Provides immediate feedback on cell viability, making it suitable for high-throughput screening.

Versatile Use—Effective for various cell types and adaptable for use in both 2D and 3D cultures.

Toxicity to Cells—Although trypan blue is not toxic to cells in the short term when used for staining, prolonged exposure can be harmful even to viable cells. Limit the exposure time to avoid altering the viability of otherwise healthy cells during analysis.

Solution Concentration—The commonly used concentration of trypan blue is 0.4%, which is optimal for visualizing cell viability without excessive dye penetration that might falsely indicate cell death.

Accurate Analysis—Ensure cells are well-dispersed to prevent clumping, which complicates accurate counting. Count cells within 5–10 minutes after staining, as trypan blue may diffuse from dead cells or stain live ones over time, leading to inaccurate results.

Trypan UltraBlue™—A higher purity version of Trypan Blue

Trypan Red Plus™—A red-colored alternative

Trypan Purple™—A purple-colored option

These stains offer reduced cellular toxicity, minimizing disruptions to cell surface receptors, including G-protein coupled receptors (GPCRs), while maintaining effective viability determination. These dyes provide enhanced flexibility and reliability in cell viability assessments.

de Oliveira V.C., et al. (2020) "Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model." PLOS ONE 15(7): e0235856. https://doi.org/10.1371/journal.pone.0235856

Del Grande, Murilo Penteado, et al. "Methylene Blue and Photodynamic Therapy for Melanomas: Inducing Different Rates of Cell Death (Necrosis and Apoptosis) in B16-F10 Melanoma Cells According to Methylene Blue Concentration and Energy Dose." Photodiagnosis and Photodynamic Therapy, vol. 37, 2022, p. 102635. ScienceDirect, https://doi.org/10.1016/j.pdpdt.2021.102635.

Jones, et al. "YAP activation is robust to dilution" Mol. Omics, 2024, 20, 554 https://pubs.rsc.org/en/content/articlepdf/2024/mo/d4mo00100a

Smith, L. A., et al. "Using Advanced Cell Culture Techniques to Differentiate Pluripotent Stem Cells and Recreate Tissue Structures Representative of Teratoma Xenografts." Frontiers in Cell and Developmental Biology, vol. 9, 2021. Frontiers, https://www.frontiersin.org/articles/10.3389/fcell.2021.667246/full. DOI: 10.3389/fcell.2021.667246.