Trypsin-EDTA solution is a reagent commonly used in cell culture for detaching adherent cells from the surface of tissue culture dishes or flasks. The solution consists of two primary components: trypsin and EDTA.
- Trypsin: Trypsin is a serine protease enzyme derived from the pancreas of animals, usually bovine or porcine. It cleaves peptide bonds in proteins, specifically at the carboxyl side of lysine and arginine residues. In cell culture, trypsin is used to break down the proteins responsible for anchoring cells to the culture surface, allowing the cells to detach.
- EDTA (Ethylenediaminetetraacetic acid): EDTA is a chelating agent that binds divalent cations such as calcium and magnesium. In cell culture, EDTA is used to chelate these cations, which are essential for maintaining the integrity of cell-cell and cell-matrix interactions. By chelating these cations, EDTA weakens the interactions and makes it easier for trypsin to detach the cells.
A common trypsin-EDTA solution is 0.25% trypsin with 0.02-0.05% EDTA in a balanced salt solution, such as phosphate-buffered saline (PBS) or Hank’s Balanced Salt Solution (HBSS). The solution is typically available as a 1x or 10x concentrated stock and should be stored at -20°C for long-term storage or 4°C for short-term use.
To use trypsin-EDTA solution for cell detachment:
- Aspirate the culture medium from the tissue culture dish or flask.
- Rinse the cell monolayer gently with PBS or another balanced salt solution to remove any residual medium.
- Add an appropriate volume of trypsin-EDTA solution to cover the cell monolayer (typically 1-2 mL for a T-25 flask or 2-3 mL for a T-75 flask).
- Incubate the cells at 37°C for a few minutes, monitoring the detachment progress under an inverted microscope.
- Once the cells are detached, add an equal volume of culture medium (containing serum or a trypsin inhibitor) to neutralize the trypsin activity.
- Gently pipette the cell suspension up and down to break up any cell clumps and transfer the cells to a new culture vessel or centrifuge tube as needed.
Always practice aseptic techniques when handling cell cultures and trypsin-EDTA solution to prevent contamination.
