Preparation of fresh solid tissue samples (enzymatic digestion)

Summary

This experiment is based on "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.

Operation method

Preparation of fresh solid tissue samples (enzymatic digestion)

Principle

There are three main effects on the dispersion of solid tissue: (1) destruction of collagen fibers and elastic fibers between tissues; (2) hydrolysis of mucopolysaccharides and other substances between tissues; and (3) hydrolysis of proteins that are tightly linked between tissue cells.

Materials and Instruments

Tissue. Enzyme.
Test tubes Centrifuge tubes Nylon mesh

Move

1. Place tissue suitable for enzymatic digestion in a centrifuge tube.

2. Add 1 to 2 ml of the selected enzyme solution to the tube containing the digested tissue.

3. Digest for 20-30 min (37°C or room temperature) with intermittent shaking or blowing.

4. Terminate the digestion, collect the cell suspension, filter through a 300-day nylon mesh to remove cell clumps, and centrifuge at a low speed to remove cell fragments.

5. The prepared single-cell suspension is fluorescently stained and assayed on a machine, or stored for later use.

Caveat

1. enzymes need to be dissolved in appropriate enzyme solutions that do not cause a decrease in enzyme potency.

2. attention should be paid to the concentration of enzyme used and the digestion time.

3. be aware of the pH of the enzyme activity; pepsin loses activity in alkaline environments and trypsin is inactive in neutral solvents.

4. pay attention to other factors affecting enzyme activity, such as the production lot number of the enzyme.

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