Dissociation of Cells from Culture Vessels with Enzyme-free Cell Dissociation Buffers

The following is a general procedure to rapidly remove various cell lines from the substratum while maintaining cellular integrity.  This procedure is not meant to be universally applicable for all cell lines.  The optimal conditions and concentrations employed for individual systems should be determined empirically.  Cell viability should be routinely monitored at the time of subculturing.  Cell viability should be greater than 90%.

  1. Warm all reagents to 37°C prior to use.
  2. Remove growth medium from cells.
  3. Thoroughly rinse cell monolayers with 5 ml Ca++- and Mg++-free PBS per T75 flask or 100 mm dish. Gently rock the flask (or dish), allowing the solution to bathe the cells for 30 to 60 seconds at room temperature. Aspirate the rinse solution and discard.
  4. Repeat step 3.
  5. Add approximately 5 ml of Cell Dissociation Buffer per T75 flask or 100 mm dish and gently bathe cells by rocking at room temperature for 1 to 2 minutes. You may check for dissociation under the microscope.  Aspirate solution and discard.
  6. Firmly tap flask or dish against palm of hand to dislodge cells. If cells do not detach quickly, allow to sit at room temperature for another 2 to 5 minutes and tap flask against palm of hand again. This may be repeated for cells that are more strongly adherent (with 5 more mls of dissociation buffer). After the cells are visibly detached add at least 5 ml of complete growth medium. Resuspend cells in growth medium.

Reference:
1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.