The chemical treatment method is to displace calcium and magnesium ions, which play an adhesive role between tissue cells, so that the cells can be dispersed. This experiment is from "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.

Operation method

Preparation of fresh solid tissue samples (chemical treatment method)

Principle

Chemical treatments are used to displace calcium and magnesium ions that act as adhesives between tissue cells, thereby dispersing the cells.

Materials and Instruments

Trypsin Tissue
EDTA
Nylon Mesh

Move

I. Preparation of reagents

1. 0.2% EDTA preparation: weigh 0.2 g of EDTA, add 100 ml of Hank's liquid, encapsulate and autoclave. Store at 0~4℃.

2. Pancreatic enzyme plus EDTA preparation: add 0.25 g of pancreatic enzyme to 200 ml of PBS (pH 7.0) with a concentration of 0.125%, and add 0.2 g of EDTA to 100 ml of PBS (pH 7.0) with a concentration of 0.2%. Take 40 ml of each mixed, divided device refrigerator storage, can be used when filtered.

Experimental method

1. Cut the tissue into thin slices and put them into test tubes.

2. Firstly, add 5 ml of EDTA liquid, 0.5 h at room temperature, centrifuge and discard.

3. Add 5 ml of trypsin-EDTA solution and incubate for 30 min at 37℃ in a constant temperature water bath shaker.

4. Filter through 300 mesh nylon mesh, centrifuge the precipitate at 1000 r/min for 5 min, and then wash with saline for 2-3 times.

5. Fix the cells or store them at low temperature.

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