Lamina Propria Mononuclear Cell Isolation: A Silica-based Density Gradient Centrifugation Technique to Obtain Mononuclear Cells from Murine Colon

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Solutions

1. As described in Table 1, prepare the Colon Buffer, Silica-Based Density Separation Media 100%, Silica-Based Density Separation Media 66%, Silica-based Density Separation Media 44%, Collagenase E Digestion Buffer, and FACS Buffer.
   1. Prepare Colon Buffer the day prior to the procedure and store overnight at 4 °C.
   2. Prepare 100% Silica-Based Density Separation Media the day prior to the procedure and stored overnight at 4 °C, placing at room temperature the morning of the procedure to thaw.
   3. Prepare 66% and 44% Silica-Based Separation Media in the morning of the isolation, using room temperature 100% Silica-Based Density Separation Media and Colon Buffer.
   4. Measure the appropriate amount of Clostridium histolyticum-derived Collagenase E and store at -20 °C overnight prior to the procedure. The following morning, dissolve in the appropriate volume of Colon Buffer to derive Collagenase Digestion Buffer. For all incubations with Collagenase Digestion Buffer on the day of the procedure, pre-warm the solutions to 37 °C.
2. For the entire of the protocol steps, keep one centrifuge at 20 °C and the rotation speed of 859 x g, with brakes inactivated (0 deceleration) for gradient centrifugation. Set another to 4 °C and rotation speed of 859 x g, with the standard deceleration, for wash steps.

2. Harvesting the Colon

1. Euthanize the mouse via CO₂ asphyxiation followed by AALAC-approved confirmatory method.
2. Place the mouse in a supine position and spray the fur with 70% ethanol. Using large tissue scissors, make a vertical midline incision and expose the intact peritoneum.
3. Using fine dissection scissors, open the peritoneum. Use forceps to move the small bowel to one side and expose the descending colon. Slightly pull upward on the descending colon to maximally expose the rectal portion of the colon. Cut the distal rectum deep in the pelvis and dissect and remove the entire colon as one unit, from the distal rectum to the cecal cap.
4. Transfer the colon in 20 mL chilled Colon Buffer in a 50 mL polypropylene tube.

3. Cleaning the Colon

1. Place the colon on a moistened paper towel and extract the solid stool by applying mild pressure to the bowel wall with the blunt end of scissors or forceps.
2. Place the colon in a Petri dish and flush the gut with 10 mL of chilled Colon Buffer using a 10 mL syringe with an 18G blunt fill needle.
3. Transfer the colon to a Colon Buffer moistened paper towel and remove the mesentery and fat with the sharp end of scissors.
4. Place the colon in a Petri dish filled with 5-10 mL chilled Colon Buffer agitating manually to wash remaining colonic contents. Repeat 2-3 times.
5. Cut the colon longitudinally from its more muscular rectal end to the proximal colon (generating a single rectangular open colon piece) in a Petri dish filled with fresh, chilled Colon Buffer. Discard existing media and refill with clean chilled Colon Buffer.
6. Wash the intestine 3 times by vigorously swirling it in the Petri dish and replacing the 5-10 mL of chilled Colon Buffer after each wash.
7. Place the rectangular colon tissue on a paper towel moistened with Colon Buffer and cut it by slicing it horizontally and then into small fragments (3 mm x 3 mm sections).
8. Collect the colon fragments carefully using fine forceps into 20 mL chilled Colon Buffer in a 50 mL polypropylene conical tube.
9. Wash the colon fragments three times, each wash in 20 mL Colon Buffer, by vigorously swirling the tube for 30 s. Between each agitation, allow the tissue fragments to settle to the bottom of the tube. Decant or vacuum aspirate the supernatant while preventing tissue fragment loss in the aspiration process between each wash.
   NOTE: There is no need to change the tube after each wash.

4. Collagenase Digestion 1

1. Add 20 mL of the Collagenase Digestion Buffer to the washed colon fragments in the 50 mL polypropylene conical tube.
2. Place the closed 50 mL tube at 37 °C in an incubated orbital shaker with the rotation rate set at 2 x g for 60 min. Ensure the tissue fragments are in constant motion during agitation; if necessary, increase the rotation rate incrementally to ensure that no tissue fragments settle to the tube bottom.

5. Prepare Silica-based Separation Media Gradients

1. Prepare 66% and 44% Silica-based Density Separation Media, using 100% Silica-based Density Separation Media at 20 °C (room temperature) and Colon Buffer.
2. Pour 5 mL of 66% Silica-based Density Separation Media into each of 3 separate 15 mL polypropylene tubes. Prepare three tubes per colon. This forms the higher density base of the gradient isolation procedure, onto which lower density separation media will be layered to create the separation gradient.
3. Store at 20 °C until use.

6. Collection of Supernatant from Digestion 1

1. Collect only the supernatant using a 25 mL serological pipette and filter the supernatant through a 40 μm-pore filtration fabric cell strainer placed into a clean 50 mL polypropylene conical tube, after Collagenase Digestion 1 is completed. Be careful not to aspirate any existing tissue fragments.
   NOTE: Retain any remaining visible tissue fragments in the tube. These will undergo second collagenase digestion (step 8).

7. Quenching Collagenase Digestion Buffer

1. Fill the 50 mL polypropylene tube completely with chilled Colon Buffer.
   NOTE: Collagenase is active at 37 °C; hence chilled buffer inactivates this enzyme.
2. Centrifuge the tube at 4 °C at 800 x g for 5 min.
   1. Discard the supernatant via vacuum aspiration. Wash cells with 25 mL of fresh Colon Buffer and centrifuge at 800 x g for 5 min.
   2. Resuspend the pellet in less than 1 mL of fresh, chilled Colon Buffer.
   3. Place the 50 mL polypropylene conical tube on ice.

8. Collagenase Digestion 2

1. Repeat step 4 (Digestion 1) with the remaining tissue fragments retained from step 6.1.

9. Tissue Disaggregation Following Digestion 2

1. Flush the tissue fragments vigorously back and forth between the tube and a 10 mL syringe through an 18G blunt-end needle.
2. Repeat this flush for a minimum of 7-8 complete passages, continuing until no gross tissue fragments or debris are visible.

10. Filter Cells

1. Pass the tissue disaggregation suspension through a 40 μm-pore filtration fabric cell strainer into a clean 50 mL polypropylene tube.
2. Wash the filtration fabric cell strainer with 10 mL chilled Colon Buffer to recover any cells ensnared in the filter.

11. Quenching Collagenase Digestion

1. Fill the 50 mL polypropylene conical tube to the rim with chilled Colon Buffer.
   NOTE: The temperature of Colon Buffer is critical to ensure quenching of collagenase activity.
2. Spin at 4 °C and 800 x g for 5 min.
3. Discard the supernatant via vacuum aspiration.
4. Wash by resuspending in 25 mL of fresh, chilled Colon Buffer, followed by centrifugation at 4 °C, 800 x g for 5 min.
5. Discard the supernatant via vacuum aspiration.
6. Pool the resuspended pellet from Collagenase Digestion 1 (step 7) to its corresponding tube from step 11.4.
7. Repeat step 11.4 (wash and centrifugation).

12. Silica-based Density Separation Media Gradient Separation

NOTE: Perform steps 12-13 as quickly as possible to ensure rapid quenching of collagenase activity.

1. Following step 11.7, resuspend each pellet in 24 mL total of 44% Silica-based Density Separation Media per colon.
2. Slowly layer 8 mL of the media from step 12.1 onto each of the three tubes prepared at step 5.2 (containing 66% Silica-based Density Separation Media), using a 10 mL serological pipette. Maintain a steady and slow flow of the 44% Density Separation Media while layering the gradient in order to avoid disruption of the interface.
3. Carefully balance all tubes within the centrifuge buckets using a weigh scale or a balance.
4. Spin the tubes 20 min at 859 x g in a centrifuge without brake at 20 °C. Allow the rotors to come to complete rest before removing the tubes, taking care not to disrupt the cells at the gradient interface.

13. Collect Mononuclear Cells from the Gradient Interface

1. Visualize the gradient interface (near the 5 mL mark), where typically a 1-2 mm thick white band (containing MNC) is present.
   NOTE: One may or may not see a white band. However, MNC will be at this interface and should cloud the clarity of the gradient interface.
2. Vacuum aspirate and discard the top 7 mL of the top gradient to allow easier pipette access to the interface.
3. Using continuous manual suction and steady rotating wrist motion, collect the interface layer of cells into a clean 50 mL polypropylene conical tube. Collect until the interface between the two gradients is clear and refractile (clear of cells).
4. Fill the collection tube with 50 mL of chilled FACS Buffer. Spin at 4 °C, 800 x g for 5 min.
5. Aspirate the supernatant via vacuum aspiration and resuspend the pellet in 1 mL of FACS Buffer.
6. Count the cells on a hemocytometer at a 1:2 dilution using appropriate dead cell exclusion methods.
7. Proceed to FACS staining or other assays with freshly isolated colonic MNC.

Table 1: Solution Preparation Table