PriCells: Normal mouse inguinal fat pad to obtain adipose-derived stem cells

Experimental Materials:
1. Hank's buffered saline solution (HBSS);
2. ASC medium: DMEM containing 10% fetal bovine serum, adding 1% P/S;
3. Collagenase solution: 100g Hank's buffered saline solution is added with 1g bovine serum albumin and 0.1g type II collagenase;
4. Polyvinylpyrrolidone iodine solution;
5. Petri dish, 9cm; centrifuge tube, 50ml; tissue culture flask, 25cm2;
6. Scissors (2), scorpion (2);
7. PBSA;
8. mice, 4-6;
9. Anesthetic: tribromoethanol;

experimental method:
1. Preheating HBSS and adipose-derived stem cell culture medium in a 37 ° C water bath;
2. Anesthetize the animal and sacrifice it;
3. Transfer the animal to the laminar flow hood;
4. 70% alcohol disinfection of the abdomen;
5. Open the abdominal cavity with a low transverse incision;
6. Use tweezers to remove the fat pad from the gonad/epigeria and groin;
7. Place the adipose tissue in a Petri dish and add HBSS;
8. After all the animal fat has been removed, transfer the fat to a new Petri dish;
9. Add HBSS containing 5% polyvinylpyrrolidone to 2-3 min;
10. Rinse the tissue and wash the fat with HBSS;
11. Transfer the fat to a 50 ml centrifuge tube using sterile forceps;
12. Shred the fat with sterile scissors;
13. Add the collagenase solution of the same volume as the tissue and mix it;
14. Place the tube in a 37 ° C water bath for 60 min, mixing occasionally;
15. Centrifugation, 50-100g, 5min;
16. Remove the centrifuge tube, shake vigorously (to completely separate the stromal cells from the primary adipocytes), and centrifuge again for 5 min;
17. Carefully remove the upper layer of fat, which contains primary fat cells, taking care not to damage the underlying matrix-vascular layer;
18. Add 5-10 ml PBSA, resuspend the pellet, and centrifuge again for 5 min;
19. Wash three times, taking care not to aspirate the matrix-vascular cell layer;
20. After the last wash, resuspend the pellet by adding 8 ml of ASC medium, transfer to a T25 flask, and place in a 37 ° C, 5% CO 2 incubator;
21. After the cells are attached to the wall for 2-4 days, change the liquid;
22. Remove the unattached cells by discarding the medium when changing the medium;
23. Change the medium twice a week later;