Isolation of Lamina Propria Cells from Mouse Small and Large Intestines

Isolation of Lamina Propria Cells from Mouse Small and Large Intestines

Modified from Miltenyi Mouse Lamina Propria Dissociation Kit 

Qing Zhao, PhD

University of Alabama at Birmingham

Required reagents and supplies

• Mouse Lamina Propria Dissociation Kit (Miltenyi, catalog # 130-097-410)
• 1x HBSS without calcium and magnesium supplemented with 10mM HEPES (HBSS w/o)
• Pre-digestion solution (prepare fresh before each experiment)
  • HBSS w/o
  • 5mM EDTA
  • 5% FBS
  • 1mM DTT
  • NOTE: warm pre-digestion solution to 37°C before use
  • NOTE: 20 mL of solution required per sample
• R10
  • 1x RPMI 1640
  • 10% FBS
  • Pen/Strep
  • .1% beta mercaptoethanol by total volume
  • 25mM HEPES 
  • NOTE: media is referred to as “RPMI complete” without 10% FBS addition
• Mesh strainers for tissues
• 100μm cell strainers
• gentleMACS C tubes (Miltenyi, #130-093-237)
• Percoll
• 10x PBS (without calcium and magnesium)
• gentleMACS Dissociator 
• Rotator

Mouse Lamina Propria Dissociation Kit Reagent preparation

1. Reconstitute Enzyme D in each vial with 3mL RPMI complete. Prepare aliquots and store at -20C. Solution is stable for 6 months after reconstitution.

2. Reconstitute Enzyme R with 2.7mL RPMI complete. Aliquot and store at -20C. Solution is stable for 6 months after reconstitution. 

3. Reconstitute Enzyme A with 1mL Buffer A. Do not vortex. Aliquot and store at -20C. Solution is stable for 6 months after reconstitution. 

LPMC Isolation Protocol

1. Remove intestinal tissue and place in cold HBSS w/o in a petri dish.

2. Clear feces from the intestines by flushing with 1x PBS using a syringe attached with a blunt-ended needle.

3. Remove Peyer’s patches (approximately 9-11 in WT mouse small intestine), cecal patch (generally at tip of cecum), and large tertiary lymphoid tissues (generally 1-2 in the colon). This will avoid contamination of the true LP population at last.

4. Remove fat tissue (important, because fat could impair the viability of lymphoid cells). 

5. Cut tissue first longitudinally, remove mucus layer with forceps, and then laterally into pieces of approximately 0.5 cm in length. Cut into smaller pieces if digesting colon. 

6. Transfer pieces into a 50 mL conical tube containing 15mls of fresh HBSS w/o and leave on ice when dissecting the next mouse. 

7. When all dissections are complete, wash tissue gently by vortexing at medium speed for 15 sec, then apply suspension to a mesh strainer, dump flow through, and replace with 20 mL pre-warmed pre-digestion solution.

Place tubes on a rack, secure with tape, and turn rack on its side. Secure rack to a rotator with tape.

8. Incubate samples for 40 minutes at 37°C under continuous gentle rotation. Set speed to medium depending on the rotator being used. 

9. After pre-digestion, vortex samples for 10 seconds and filter suspensions through mesh strainer, dump flow through (this contains the gut epithelial cells). 

10. Transfer the tissue pieces into a new 50 mL tube with 20 mL pre-warmed HBSS w/o.

11. Incubate for 15 minutes at 37C on the rotator. 

In the meantime, prepare the digestion solution. 

• Prepare 2.5 mL of digestion solution per sample, containing 80μl of Enzyme D, 40μl of Enzyme R, and 10μl of Enzyme A. Make up the rest with pre-warmed R10. 

12. Vortex the samples for 10 seconds and filter suspension through the mesh strainer. 

13. Transfer the tissue pieces into a gentleMACS C tube and add prewarmed 2.5 mL digestion solution. Mix gently and close the tube tightly. 

14. Place C tubes on a rack, secure with tape, and turn rack on its side. Make sure the round side of the C tubes is facing downward on the rack. Secure rack to a rotator with tape. Incubate for 30 minutes at 37C on the rotator.  Set speed to slightly higher than the pre-digestion step. 

15. After digestion, make sure that C tubes are closed tightly. Attach the C tube upside down onto the gentleMACS dissociator. 

16. Run the program m_intestine_01 twice for small intestine and three times for colon.

17. Detach C tube from the dissociator and spin at room temperature for 4 minutes at 300g.

In the meantime, prepare Percoll solutions:

100% Percoll = 10 mL 10x PBS + 90 mL stock Percoll (prewarmed) 

75% Percoll = 15 mL 100% Percoll + 5 mL 1x PBS 

40% Percoll = 16 mL 100% Percoll + 24 mL pre-warmed R10

NOTE: 2 mL of 75% and 5 mL of 40% are needed per sample, so modify volumes accordingly (prepare 3.5mL of 100% Percoll for each sample).  

18. Resuspend the digested sample with 5 mL cold RPMI complete and apply the suspension to a 100μm cell strainer on a new 50 mL tube for each sample.

19. Keep the strainer on the 50 mL tube. Wash C tube with cold RPMI complete and apply to strainer. Then wash strainer with additional cold RPMI complete. The flowthrough contains intestinal LPMCs. There should be about 15-20 mL of RPMI complete in the 50 mL tube. 

20. Centrifuge 50 mL tubes at room temperature for 8 minutes at 300g.  Remove supernatant carefully (tissue could be loosely aggregated at the bottom of the tube).

21. Resuspend cells in 5 mL 40% Percoll.  Then place resuspended samples on top of 2 mL 75% Percoll in a 15 mL tube. Add the top layer very slowly, being careful not to mix the 2 layers. The red layer from the 40% and the clear layer from the 75% should be distinct. 

22. Centrifuge at room temperature (important) for 25 minutes at 800g with no brake. 

23. Remove the fat layer at the top first, then transfer the mononuclear cell layer (middle layer) to a new 15 mL tube with transfer pipet. Add cold RPMI complete and centrifuge 8 minutes at 4C at 300g.

24. Resuspend cells in cold R10. Expected yield from a naive adult WT C57BL/6 mouse: around 5 million from the small intestine and around 1 million from the colon.

25. Stain cells immediately or store cells in R10 on ice overnight for staining the next day.