0.25% Trypsin-EDTA (1x), phenol red consists of dried trypsin powder (a mixture of proteases from porcine pancreas, irradiated and sterilized) dissolved in EDTA. Trypsin hydrolyzes intercellular proteins and disperses primary tissues or adherent cells into individual cells, and can be widely used for cell dissociation, cell culture passaging and primary tissue dissociation.
Cell dissociation:
1. Aspirate the culture medium and wash the cells twice with sterile PBS, Hanks or serum-free culture medium to remove residual serum.
2. Add the pre-warmed trypsin-EDTA dissociation solution to cover the cells and incubate at room temperature for approximately 2 minutes. Note that the actual incubation time varies with the cell line used.
3. Observe under the microscope, and when ≥90% of the cells are detached, terminate the dissociation by adding twice the volume of serum-containing complete medium.
4. Centrifuge at 200×g for 5-10 minutes and resuspend the cell precipitates in complete medium containing serum for subsequent experiments.
Tissue dissociation:
Determine the optimal conditions based on relevant experience and literature, as the digestion time required varies from different tissues.
