Trypan Blue (Direct Blue 14) | CAS 72-57-1 | Dye Content ≥70%

Trypan Blue Exclusion Cell Viability Assay

• Trypan Blue is the standard reagent for the dye exclusion assay, the most widely used method for determining cell viability in culture. The assay exploits the principle that viable cells with intact plasma membranes exclude the negatively charged dye, while cells with compromised membranes allow entry and become stained blue. In the standard protocol, a 0.4% Trypan Blue solution is mixed 1:1 with the cell suspension and loaded onto a haemocytometer or automated cell counter within 1–5 minutes. Unstained (viable) and blue-stained (non-viable) cells are counted to calculate the percentage viability. This method is applied routinely in cell culture subpassaging, cytotoxicity screening, post-thaw viability assessment, and quality control of primary cell isolations.
• Strober (Curr. Protoc. Immunol. 2001, Appendix 3B) established the trypan blue exclusion test as the standard reference protocol for assessing cell viability, defining the methodology adopted universally in cell biology laboratories.

Automated Cell Counting and High-Throughput Viability Assessment

• Trypan Blue is the preferred dye for automated cell counters, which use brightfield image analysis to distinguish blue-stained non-viable cells from unstained viable cells. Instruments such as the Countess, TC20, and Vi-CELL systems are calibrated specifically for Trypan Blue contrast, enabling rapid and reproducible viability measurements across large sample numbers. The compatibility of Trypan Blue with automated counting platforms makes it essential for standardised cell counting in Good Manufacturing Practice (GMP) environments, bioprocess monitoring, and high-throughput drug screening workflows where consistent and operator-independent viability data are required.

Mycorrhizal Root Colonisation Staining

• Trypan Blue is used as a non-toxic alternative to chlorazol black E and acid fuchsin for staining arbuscular mycorrhizal (AM) fungal structures within plant roots. In the standard protocol, roots are cleared with potassium hydroxide, acidified with hydrochloric acid, and then stained with 0.05% Trypan Blue in lactoglycerol. The dye selectively stains fungal hyphae, vesicles, and arbuscules within root cortical cells, enabling quantification of mycorrhizal colonisation rates by the gridline intersect or magnified intersection methods. This staining approach is widely adopted in soil ecology and plant biology for studying symbiotic interactions between AM fungi and host plant root systems.
• Phillips and Hayman (Trans. Br. Mycol. Soc. 1970, 55, 158) developed the lactophenol trypan blue staining method for mycorrhizal root colonisation, which was subsequently adapted to the less toxic lactoglycerol-based protocol now in standard use.

Ophthalmic Surgical Stain for Capsular and Membrane Visualisation

• Trypan Blue is approved as an ophthalmic vital dye for staining the anterior lens capsule during cataract surgery, particularly in cases of mature or hypermature cataracts where capsular visualisation is poor. A 0.06% Trypan Blue solution is injected under an air bubble into the anterior chamber, where it selectively stains the anterior capsule blue, enabling the surgeon to perform a controlled continuous curvilinear capsulorhexis. Trypan Blue is also used to stain epiretinal membranes and internal limiting membranes during vitreoretinal surgery, facilitating membrane peeling and improving surgical outcomes in macular hole and epiretinal membrane repair procedures.

Cell Membrane Permeability and Cytotoxicity Studies

• Trypan Blue uptake serves as a quantitative endpoint in cytotoxicity assays measuring the membrane-damaging effects of chemical, physical, and biological agents on cultured cells. The rate and extent of dye uptake correlate with the degree of membrane disruption, enabling dose-response characterisation of cytotoxic compounds, pore-forming toxins, and physical treatments such as electroporation or freeze-thaw cycling. Trypan Blue-based cytotoxicity measurements complement metabolic activity assays such as MTT and resazurin reduction by providing a direct readout of membrane integrity rather than metabolic function, which is particularly important for distinguishing between cytostatic and cytolytic mechanisms of cell death.
• Chan et al. (PLoS One 2020, 15, e0227950) characterised the morphological effects of trypan blue on dead and dying cells, establishing quantitative criteria for distinguishing membrane rupture from early-stage permeabilisation in dye exclusion assays.