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

To begin, add 20 milliliters of collagenase digestion buffer to the tube containing the washed colon fragments. Seal the tube and place it in an orbital shaker at 37 degrees Celsius with a rotation rate of 2 x g for 60 minutes. Ensure the tissue fragments are in constant motion during agitation.

Pour 5 milliliters of 66% silica-based density separation media into each of three separate 15-milliliter polypropylene tubes. Next, use a 25-milliliter serological pipette to collect only the supernatant from Collagenase Digestion 1. Filter the supernatant through a 40-microliter-pore filtration fabric cell strainer placed into a clean 50-milliliter polypropylene conical tube. Fill the tube completely with chilled colon buffer to quench the collagenase digestion buffer. Then, spin the cells, aspirate the supernatant, and keep the pellet on ice.

Continue to perform Collagenase Digestion 2 as outlined in the text protocol. After Collagenase Digestion 2 is completed, flush the tissue fragments vigorously back and forth between the tube and a 10-milliliter syringe through an 18G blunt-end needle. Repeat this flush for at least seven to eight passages, continuing until no gross tissue fragments or debris are visible.

Next, pass the tissue disaggregation suspension through a 40-micrometer-pore filtration fabric cell strainer placed into a clean 50-milliliter polypropylene tube. Fill the tube to the rim with chilled colon buffer to quench the digestion and centrifuge at 800 x g and at 4 degrees Celsius for 5 minutes.

Discard the supernatant via vacuum aspiration and pull the resuspended pellet from Collagenase Digestion 1 to its corresponding tube. After this, resuspend each pellet in 24 milliliters of 44% silica-based density separation media per colon. Use a 10-milliliter serological pipette to slowly layer 8 milliliters of this media onto each of three tubes containing 66% silica-based density separation media.

Carefully balance the tubes within centrifuge buckets using a weigh scale or a balance. Centrifuge at 859 x g and at 20 degrees Celsius for 20 minutes without the break. Allow the rotors to come to complete rest before removing tubes, taking care not to disrupt the cells at the gradient interface.

Visualize the gradient interface near the 5-milliliter mark where typically a 1-2 millimeter thick white band is present. Vacuum aspirate and discard the top 7 milliliters of the gradient to allow easier pipette access to the interface. Using continuous manual suction and steady rotating wrist motion, collect the interface layer of cells. Collect until the interface between the two gradients is clear and refractile, and transfer the interface to a new 50-milliliter polypropylene conical tube.