Dissociation Enzyme Solution III

This sterile-filtered digestion solution is ready-to-use for adherent cell culture dissociation and selected tissue digestion. It offers convenient and rapid processing, typically detaching most adherent cells within approximately 1 minute at room temperature. Cells may be collected as soon as obvious morphological changes are observed, without waiting for complete rounding.

(1) Adherent cell dissociation: 1) Aspirate culture medium and wash cells once with sterile PBS, Hanks' solution, or serum-free medium to remove residual serum. 2) Add a small volume of Trypsin-EDTA Cell Dissociation Solution (with phenol red) sufficient to cover the cells, incubate at room temperature for 30 seconds to 2 minutes. Different cell types require different incubation times. 3) Monitor under microscope; when cells show obvious contraction and appear detached from the culture vessel bottom, or can be easily dislodged by pipetting, remove the dissociation solution. Add complete medium containing serum, pipette to collect cells for downstream use. 4) If digestion is insufficient, re-apply dissociation solution. If digestion is excessive and cells have begun to detach spontaneously, collect all cells with complete medium, centrifuge at 200×g for 5 minutes, remove dissociation solution, and resuspend in complete medium for subsequent use. (2) Tissue digestion: Different tissues require significantly different digestion times; digest until tissue can be adequately dispersed.