小鼠原代脂肪细胞的分离培养方法

小鼠原代脂肪细胞的分离培养方法

Materials and Solutions HEPES Buffer (pH 7.4) Containing:

1. 5 mM D-glucose

2. 2% BSA

3. 135 mM NaCl

4. 2.2 mM CaCl2.2H2O

5. 1.25 mM MgSO4.7H2O

6. 0.45 mM KH2PO4

7. 2.17 mM Na2HPO4

8. 10 mM HEPES

Type 1 collegenase solution

Dissolve 1.25 mg collegenase type 1 powder in 1 ml of HEPES buffer

1% FBS-DMEM media

Add 5 ml FBS in 500 ml bottle of DMEM media

Procedure

1. Sacrifice animals by appropriate killing method. Dissect epididymal

fat depots.

2. Mince fat tissues with scissors in HEPES buffer.

3. Digest adipose tissue fragments in the HEPES buffer with Typ1

collegenase at 370C with gentle shaking for 30 min.

4. Dilute the resulting cell suspension in HEPES buffer.

5. Filter diluted cell suspension with 400 μm nylon mesh to remove

undigested tissues.

6. Wash filtrate three times with HEPES buffer.

7. Suspend isolated adipocytes in 1% FBS-DMEM and incubate at

370C for 40 min.

8. Plate isolated adipocytes (150 μl of 2:1 ratio of packed cells to

medium) were then plated on 500 μl of collegen matrix in 6 well culture plates.