DAY 1:
[A.] Prepare Reagents

  1. Wash Buffer: 5% FBS/F12
    25 ml FBS
    5 ml AA
    475 ml DMEMF12 or HamF12
    500 ml
    Filter sterilize.
  2. 0.25% Trypsin-EDTA (InVitrogen)
  3. Growth media:
    250 μl 1mg/ml insulin (5μg/ml final)
    50 μl 1mg/ml hydrocortisone (1μg/ml final)
    25 μl 10μg/ml EGF (5ng/ml final)
    250 μl 10mg/ml gentamycin (50μg/ml final)
    500 μl antifungal/antibiotic
    2.5 ml FBS
    47 ml HamF12
    50 ml
    Filter sterilize.
  4. Digestion Media
    250 μl AA
    25 mg collagenase (1mg/ml)
    24.75 ml HamF12
    25 ml
    Filter sterilize and prepare fresh for each use.

[B.] Isolate MECs from mice

1. For the glands of 3 mice, add ~20ml of wash buffer to a 50ml conical tube.

2. Weigh tube with wash buffer and record. Place on ice.

3. Proceed with dissection, removing the #3, 4, 5 glands, removing the lymph node. Transfer glands to wash buffer on ice.

4. Weigh tubes with tissue, and subtract values to determine grams of tissue.

5. Chop tissue till very pasty—no clumps.

6. Add the appropriate amount of digestion buffer to each tube: 10 ml/gram of tissue

7. Incubate tissue at 37°C on rocker, at ~50 rpm for about 14 hours. (Place tubes at a 45° angle.)

DAY 2:

8. After digestion is complete, shake digested cells vigorously to loosen cells from fat.
Transfer cells to a 15ml conical tube.

9. Spin at 1000 rpm at for 10′ using a swing bucket rotor at RT.
Carefully aspirate supernatant.

10. Resuspend cells in 2ml of 0.25% trypsin-EDTA by pipetting up and down.
Incubate at RT for 5′, gently inverting throughout the incubation.

Note: A large fibrous mass will ppt when trypsin is added. Also, the volume of trypsin will vary depending on the size of the pellet. After a few minutes, the media should appear clear rather than thick and clumpy.

11. Neutralize trypsin with 5ml of wash buffer.Pass cells through a 70μm filter into a 50ml conical tube. (filtration will get rid of the ppt)

12. Transfer cells back to a 15ml conical tube and bring the volume to 15ml with wash buffer.
Spin at 1000 rpm for 10′.
Carefully aspirate supernatant.

13. Continue washing to ensure that enzymatic activity is stopped (~3-4 times total). Each time, spin at 300 × g for 5′ at RT.

14. Resuspend pellet in 3ml of growth media. Dilute cells 1:10-1:50 and count on a hemacytometer. Yield: ~2.5 × 106 cells per 0.5 g tissue