Isolation of cells and FACS + Culture medium of McSC

Isolation of cells and FACS

For the isolation of telogen cells

1. Kill the mice with CO₂.

2. Shave the dorsal hair of the mouse. Keep the hair as short as possible.

3. Cut the skin off the back of the mouse. Place the skin on the foil (dermis up).

4. Use the scalpel to scrape subcutaneous fat from the dermis. 

5. Float the skin (with the dermis side facing down) on 10 ml 0.25% trypsin.

6. Cut the skin into 1 cm wide strips.

7. Incubate skin in 37 °C, 80 r/min. Depend on the age and sex of the mice: 20 days of mice (30 minutes); 50-60 days of female mice (30 minutes); 50-60 days of male mice (45 minutes).

8. After incubation, neutralize the trypsin with 10 ml of PBS containing 5% decalcified serum.

9. Gently scalp from the epidermis side to isolate epidermal cells.

10. Pipette up and down for several times to dissociate cells.

11. Filter through 70um and 40um cell strainer.

12. 300g centrifuged for 15 minutes to collect the cells.

13. Remove the supernatant.

14. Use 400 ul PBS with 5% serum to resuspend the cells.

15. Transfer the cells to 5 ml flow tube.

16. Add the 1st antibody, incubate at 4 °C for 15 to 20 minutes. 

17. Mix two or three times during the incubation period.

18. Add 4 ml PBS with 5% serum.

19. Centrifuge for 5 minutes at 300 g to remove the supernatant;

20. If the second antibody is need, repeat Step 16-19.

21 Finally, add DAPI to the cell suspension at a final concentration of 0.2 μg/ml to remove dead cells.

19) Sort the target cell population according to the markers.

For the isolation of anagen cells

1. Kill the mice with CO₂.

2. Shave the dorsal hair of the mouse. Keep the hair as short as possible.

3. Cut the skin off the back of the mouse. Place the skin on the foil (dermis up).

4. Use the scalpel to scrape subcutaneous fat from the dermis.

5. Float the skin (with the dermis side facing down) on 10 ml HBSS with collagenase type 1.

6. Cut the skin into small pieces.

7. Incubate skin in 37 °C, 80 r/min. 

8. During incubation, scrape the digested portion of the dermis with a scalpel every 30 minutes to expose the undigested portion.

9. After 1.5 to 2 hours of incubation, the entire skin is fully digested.

10. All tissues are collected and transferred to a 50 ml centrifuge tube.

11. Supplemented with PBS with 5% serum to 50 ml.

12. 300g centrifuge for 15 minutes.

13. Place the pellet in 10 ml 0.25% trypsin. Shake in a 37 °C for 30 minutes.

14. Neutralization with PBS with 5% serum

15. Pipette up and down for several times to dissociate cells.

16. Filter through 70um and 40um cell strainer.

17. 300g centrifuge for 10 minutes to remove the supernatant.

18. Use 400 ul PBS with 5% serum to resuspend the cells.

19. Transfer the cells to 5 ml flow tube.

20. Add the 1st antibody, incubate at 4 °C for 15 to 20 minutes. 

21. Mix two or three times during the incubation period.

22. Add 4 ml PBS with 5% serum.

23. Centrifuge for 5 minutes at 300 g to remove the supernatant;

24. If the second antibody is need, repeat Step 20-23.

25 Finally, add DAPI to the cell suspension at a final concentration of 0.2 μg/ml to remove dead cells.

19) Sort the target cell population according to the markers.

Culture medium of McSC

1. 50mL RPMI 1640 Phenol Red-free.
2. Add 245 μL 3mg/ml Phenol Red.
3. 5.6 mL FBS.
4. 560 μL 100x P/S.
5. Add 400nM TPA、20ng/mL SCF、20nM ET1、400pM CT

TPA (12-O-tetradecanoylphorbol-13-acetate): 400nM

SCF (Stem cell factor): 20ng/mL

ET1 (Endothelin 1): 20nM

CT (Cholera toxin): 400pM

Used in 2 weeks.