trypsynaEDTA

Trypsin-EDTA is a commonly used enzymatic solution in mammalian cell culture to detach adherent cells from culture vessels. It combines trypsin, a serine protease, with EDTA, a chelator of divalent cations. Trypsin cleaves peptide bonds in proteins involved in cell–substrate and cell–cell adhesion, while EDTA chelates calcium and magnesium ions that stabilize those adhesion proteins, facilitating detachment. Typical formulations use a low concentration of trypsin in a calcium- and magnesium-free buffer, often with EDTA; commercial products vary in base solution and concentrations.

Usage involves washing cells and exposing them to trypsin-EDTA for a short period (commonly 2–10 minutes) at 20–37°C until cells detach. The enzymatic reaction is usually neutralized with serum-containing medium or a specific trypsin inhibitor, after which the detached cells are collected by gentle pipetting and reseeded. It is essential to minimize exposure time to preserve cell viability and surface proteins; excessive exposure can damage receptors and affect downstream experiments.

Considerations include cell type sensitivity to proteolysis; some cells respond poorly to trypsin and may require shorter exposure or alternative dissociation methods such as enzyme-free buffers, collagenase, or Accutase. Trypsin-EDTA may alter surface markers, which should be considered for experiments involving cell surface proteins.

Safety and storage: trypsin-EDTA is handled as a biohazardous enzyme preparation and should be used with appropriate protective equipment. Stock solutions are typically stored at low temperatures (often -20°C) in aliquots to avoid repeated freeze-thaw cycles.