Isolation of lymphocytes from mouse skin

Isolation of lymphocytes from mouse skin 

Procedure

Ears: 

• Cut off non-tagged ear (can use independently or with trunk skin as desired) 

Trunk skin:

• Shave area of skin you want to harvest (i.e. back). 2x3cm piece of back skin should be sufficient for 1-2 flow panels.
• Cut out pieces of trunk skin as desired. No size limit per mouse. Note anagen areas will have an altered immune compartment and be more difficult to isolated cell suspension. 
• Gently scrape off subcutaneous fat using forceps
• Add 1mL of digestion cocktail to skin  

1. Chop skin very finely with scissors for 1-2 min until you no longer feel any resistance (create a “paste”)
2. Put skin into 50 ml conical tube with digestion cocktail (should use 3-5mL total for entire trunk skin; 1.5mL for 2cm² patch)
3. Incubate in heated shaker @37°C for 45min 
4. Add 15mL of cold C10 media to each tube and immediately place on ice; proceed quickly to avoid prolonged time in digestion cocktail.
5. Vortex tubes for 15 seconds
6. Pour through a 40um filter (if only interested in lymphocytes, otherwise use 100um) filter into 50mL conical 
7. Spin down, count, (may need to refilter), FACS, Re-plate for i.c. cytokine stain

Digestion cocktail

Make 330uL aliquots in HBSS and freeze for future usage: 

collagenase XI (Sigma, # C9407): 60 mg/mL

hyaluronidase (Sigma, # H3506): 15 mg/mL

DNase (Sigma, DN25): 3mg/mL

To make working stock add 330uL of each enzyme aliquot to 9mL of C10 media. 

C10: Medium:                                                           ml

RPMI 1640                                                                500

10% Calf Serum (heat inactivated)                           60

1% Pen Strep                                                           5

1 mM Na-pyruvate (100mM Stock)                          5

1% Hepes (1M Stock)                                              5

1x non essential aa (100x)                                       5

* BME                                                                       3

* For BME, take 78 ul of 98% BME from the stock and add it to 100 ml of PBS.  Use 3 ml of the solution to make C10.

Skin Epithelial-Dermal Separation Method

Materials

-C10 media (Complete RPMI)

-skin digestion cocktail

-0.5% Trypsin (no EDTA)(Gibco, cat# 15090-046)

OR

-Thermolysin in PBS, 0.25 mg/ml (Sigma, cat# T7902)

-6 well plate(s)

-60mm petri dishes

-100 um cell strainer

-50 ml conicals

Method:

1. Put 2-3 ml of trypsin or thermolysin in each well of a 6 well plate.

2. Sacrifice mice and shave dorsal back skin. (rinse with EtOH)

3. Pin down skin, epidermal side down/dermal side up, at four corners.

4. Measure out a 1.5 x 1.5 cm or 2 x 2 cm area of skin and cut a square with scissors. 

5. Pin down four corners of the square, still keeping the epidermal side down, dermal side facing up.

6. Use one forceps to hold skin still, and use other forceps to scrape off subcutaneous fat.

7. If desired, put fat in 50 ml conical with 1 ml C10 media and place on ice.

8. Float de-fatted skin in well containing trypsin or thermolysin, dermal side down, epidermal side up. Make sure sides are not curled up.

9. Place in mouse incubator for 45 minutes.

10. Remove 6 well plate from incubator.

11. Place 1 ml of C10 media in the lid of a 60mm petri dish.

12. Remove skin from 6 well plate and float it on the C10 media in the petri dish, with the epidermal side up. 

13. Using two forceps again, gently scrape off the epithelium from the dermis into the C10 media (media should become a little cloudy; using too much force when scraping will likely liberate dermal components in the media)

14. If dermis is desired, rinse it with 1 ml C10 media and place in lid or bottom of another petri dish.

15. Mince fragments of epithelium as best as you can using scissors (some protocols place the C10 media containing the epithelial fragments in a syringe and plunge several times to fragment, but I’ve never attempted this). 

16. Cut small portion of distal tip off P1000 tip. Pipette C10 media containing epithelial fragments up and down several times to liberate additional cells. 

17. Pipette C10 media containing epithelial fragments through 100 um cell strainer over a 50 ml conical. Wash with additional C10 media.

18. Spin down at 1500 rpm for 5 minutes and count.

19. If downstream analysis of dermal and subcutaneous cells is desired, then mince dermis and subcutanoues fatty tissue with scissors, place into 50 ml conical, and add skin digestion media at half the normal concentration.

20. Incubate dermal prep in 37 degree bacterial shaker for 25-30 min. Then proceed with the skin FACS protocol.

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