Single-cell tissue preparation

Isolation of mouse thymic epithelial cells by enzymatic digestion and density gradient enrichment

Materials

60 mm Petri dishes

Razor blades

Glass Pasteur pipettes (9 inches w/ cotton plug)

15 and 50 mL conical tubes

70μm cell strainers

Plastic transfer pipettes

Pipette-Aid

Swinging bucket centrifuge (e.g. Eppendorf 5810R)

DMEM (high glucose)

Fetal bovine serum (FBS)

DNase 1 (Roche, 10104159001)
    - Reconstitute with 1 ml water to yield 100 mg/ml stock

Liberase TM (Roche, 5401127001)
    - Reconstitute one 50 mg bottle with 10 mL water to yield 5 mg/mL
    - Gently shake while on ice for ~10-15’
    - Freeze 288 L or 576 L aliquots at -20°C for future use

MACS Buffer (5g BSA, 4mL 0.5M EDTA, 10x PBS Ca/Mg free, milliQ to 1L)

Percoll Plus (Cytiva, 17544501)

Protocol

Tissue Digestion

1. Prepare 2% FBS in DMEM
   - Add 10 mL FBS to 500 mL media (good for < 1month at 4°C if maintained sterile)
2. Prepare digestion medium (see below)
   - Add Liberase and DNase to appropriate volume of DMEM/FBS)
   - Place digestion media in a 37°C water bath to warm
   - Each digestion requires 12 mL of media
   - If anticipating an extra round of digestion (e.g. if pooling >3 thymi per digestion tube) scale up accordingly

Digestion Media Composition

3. Make MACS Buffer (for 1L): 
    -4 mL 500 mM EDTA
    - 100 mL 10X PBS
    - 900 mL milliQ H2O
    - 5 g BSA

4. Pipette 5 mL cold 2% FBS/DMEM into a 60 mm Petri dish; one for each thymus or for each digestion if pooling thymi

5. Harvest thymi into Petri dishes on ice

6. Mince thymi with a new, clean razor blade
    - Generally, change razor blades between samples unless minor cross-contamination is acceptable

7. Transfer minced tissue and all medium into a 15 mL conical tube for each different sample with a glass Pasteur pipette connected to a pipette-aid

8. Wash Petri dish with 5 mL 2% FBS/DMEM and transfer wash to tube from step 6

9. Firmly vortex each 15 mL tube for 5 seconds

10. Place tubes back on ice and incubate for 3 minutes or until fragments have settled to the bottom of the tube

11. Remove and discard as much supernatant as possible without disturbing the settled tissue fragments

12. Add 4 mL digestion medium to each tube and forcefully mix 10 times by pipetting fragments through a glass Pasteur pipette using a Pipette-Aid
   - Leave the glass Pasteur pipette in the tube

13. Incubate tubes for 4 minutes in a 37°C water bath

14. Forcefully mix 10 times with the glass Pasteur pipette

15. Incubate tubes for 4 minutes in the 37°C water bath

16. Forcefully mix 10 times with the glass Pasteur pipette

17. Briefly pulse tubes in a centrifuge to draw tissue fragments to bottom of tube
    - Bring up to ∼ 1500 rpm in a swinging bucket centrifuge and stop

18. Remove as much supernatant as possible without disturbing the tissue pellet and add it to a 50 mL conical tube containing 20 mL MACS buffer on ice
    - This solution stops the digestion for the transferred supernatant

19. Add 4 mL digestion medium to each tube and REPEAT STEPS 12-18 for 2 additional full rounds of digestion or UNTIL MEDIA IS ALMOST CLEAR AFTER MIXING
    - May add a fourth round if digesting multiple pooled thymi (3-5 per tube) together

20. Add all remaining fluid from digestion tube to the 50 mL conical with MACS 
    - Use 5 mL of solution from 50 mL conical tube to rinse the inside of the 15 mL digestion tube and add the wash back to the 50 mL conical

21. Spin tube at 1250 rpm for 8 minutes to pellet cells

22. Check for a pellet and dump supernatant

23. Gently resuspend the pellet in 20 mL MACS buffer to fully quench the enzyme and wash the cells
    - IMPORTANT: Use a 1 mL pipette to resuspend the pellet in 1 mL MACS buffer before topping up with 19 mL
    - DO NOT VORTEX (will result in cell clumps)

24. Incubate on ice for 10 minutes to completely quench the digestion

25. Spin suspension at 1250 rpm for 8 minutes to pellet quenched cells

Percoll Cell Separation

26. Dilute Percoll
    - Need ~4 mL of 1.124 Percoll for heavy (1.115) and ~2 mL for light (1.065) per sample
    - Be sure to dilute Percoll stock using 10X PBS and heavy and light using 1X PBS

Percoll densities

27. Check for a pellet, dump supernatant, and gently resuspend in 4 mL heavy (1.115) Percoll
    - Use a 1 mL pipette to resuspend, DO NOT VORTEX (will result in cell clumps)

28. Pipette 1 mL FBS into a new 15 mL conical tube for each sample
    - Cap conical, vortex top and bottom, and swish FBS around inside to coat interior surface of tube and 
    - Tap out residual FBS on a paper towel

29. Use a plastic transfer pipette to strain suspension from step 37 through a 70 m cell strainer and into the 15 mL conical from step 38 
    - Wipe bottom of strainer on edge of conical to capture droplet of cells stuck to strainer mesh

30. Set up gradient apparatus by tilting a foam 15 mL conical tube holder at 45-60 degrees, leaning it against an ice bucket, and securing it with doughnut weights

31. Set a Pipette-Aid to “slow” and gently layer 2 mL light (1.065) Percoll onto the heavy fraction

32. Layer 2 mL of 1X PBS onto the light Percoll layer

33. Check for 3 crisply layered fractions by looking through the side of the tube

34. Spin tubes for 30 minutes (Eppendorf 5810R swinging bucket centrifuge)
    - 2700 rpm
    - 4°C
    - Brake set to 0 and the acceleration set to 5 
    - Spin will take about 45 minutes

35. Collect the mTEC-enriched fraction (~3 mL) (upper layer, first interphase, and 1/3 of light Percoll) using a transfer pipette and move to 15 mL conical
    - The cells are layered at the interphase but it is easiest to take some of the liquid above and below, as described 

36. Top volume up to 10.6 mL with PBS (estimated from the side of the 15mL tube)

37. Mix by inverting and then move 600 L

38. Count cells if desired
    - Should have ~3 million per 4-6 week old thymus (varies by age and strain)

39. Spin at 1250 rpm for 5 minutes to pellet

40. Carefully aspirate supernatant and resuspend cells for downstream applications
    - Move cells to U-bottom plate if preparing for flow cytometry