✓Perform enzymatic and mechanical digestion of mouse salivary gland tissue
✓Isolate and culture salispheres containing stem cell populations
✓Identify stem cell characteristics using phase-contrast microscopy and immunostaining
Protocol
An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.
Difficulty
intermediate
Total time
~4–6 hours per mouse (tissue harvest to plating); sphere formation assessment over 3–7 days
Model organism
Mouse
Biosafety
BSL-1
Steps
1
Digest salivary gland tissue mechanically and enzymatically
Extract mouse salivary gland tissue and perform combined enzymatic digestion and mechanical trituration to dissociate the tissue into single cells and small cell aggregates.
▶ 01:32
2
Prepare digested salivary gland tissue for plating
Process the enzymatically digested tissue by washing, filtering, and centrifugation steps to obtain a clean cell suspension ready for culture.
▶ 05:00
3
Plate salivary gland cells in culture medium
Seed the isolated cell suspension into appropriate culture dishes or wells at optimal density to allow sphere formation and stem cell enrichment.
▶ 07:32
4
Examine salispheres and cells by microscopy and staining
Visualize developing salispheres using phase-contrast microscopy and apply immunostaining to confirm stem cell markers and characterize cell populations.
▶ 08:39
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