Establishment of primary intestinal epithelial cells and leukocytes from the three-spined stickleback, Gasterosteus aculeatus

After following approved euthanasia procedures, place the fish’s body On ice.

Make a ventral incision from the cloaca to the jaw using sharp surgical scissors.

Make two lateral incisions just behind the opercular flaps down to the lateral line of the fish.

Using two pins, secure the fish on its dorsal side on a dissecting pad.

To detach the intestine, make a cut at pyloric caeca on one side and the cloaca on the other side.

Place intestine into a Petri dish containing 10mL of cold 1X PBS.

Using forceps and mini dissecting scissors, open the intestine by making a longitudinal incision.

Bring the Petri dish containing the intestine into the biological safety cabinet and wash the intestine by submerging in two successive 0.1% povidone-iodine washes for 0h 5m 0s each.

Wash twice for 0h 5m 0s each in a Petri dish containing 10mL of cold 1x PBS to remove iodine.

Transfer opened gut in 10mL of mucus removal solution and incubate for 0h 10m 0s at 17°C on a gyratory rocker for 0h 10m 0s.

Resuspend the gut tissue in a fresh 10mL of mucus removal solution and incubate again incubate for 0h 10m 0s at 17°C on a gyratory rocker.

Transfer the gut tissue to 10mL of epithelial cells recovery solution and incubate for 0h 10m 0s at 17°C on a gyratory rocker.

In order to recover epithelial cells in the suspension, remove the gut tissue from Epithelial cells recovery solution and keep it On ice for enzymatic digestion step. Centrifuge the cell suspension at 300x g,0h 0m 0s for 0h 10m 0s at 17°C.

Remove the supernatant and resuspend the epithelial cell pellet in HBSS with 2% FBS and 1% Pen Strep.

Transfer the intestinal tissue from step 13 to 7mL of enzymatic digestion solution, then incubate for 0h 30m 0s at 17°C on a gyratory shaker.

Collect and save the cell suspension at 17°C.

Resuspend the remaining intestinal tissue removed from the enzymatic digestion solution in 7mL of fresh enzymatic digestion solution for a second enzymatic digestion.

Incubate for an additional 0h 30m 0s at 17°C on a gyratory shaker.

Recover the cell suspension and pool with cell suspensions obtained from step 16 in a 15 ml conical tube and keep at 17°C.

Filter the obtained cell suspension through a 40 µm mesh cell strainer into a new tube to remove cell clumps.

Ccntrifuge the obtained unicellular suspension at 300x g,0h 0m 0s for 0h 10m 0s at 17°C.

Resuspend the cell pellet in 5mL L15 with 2% FBS and 1% pen Strep.

Use double-density leukocyte isolation medium to recover all leukocytes from the cell suspension.

In a 15 mL conical tube, add 10mL of the density medium.

Carefully layer 5mL of the cell suspension onto the density medium and mix the two phases.

Centrifuge for 0h 20m 0s at 750x g,0h 0m 0s at 17°C.

After the density centrifugation, one white layer of cells appears between the L15 medium and Ficoll. Aspirate the top layer of the L15 medium.

Next, transfer the mononuclear and polymorphonuclear cell layer to a new conical tube, while making sure to not aspirate the Ficoll gradient with the cells. Wash the cells by centrifuging them at 17°C, 300x g,0h 0m 0s, once with 10mL of L15 2% FBS and 1% PenStrep.

Seed the cells into 96 wells plate at a density of 1x10⁶ cells/ml in L15 media with 10% FBS and 1% PenStrep.