TITLE: Establishment of primary intestinal epithelial cells and leukocytes from the three-spined stickleback, Gasterosteus aculeatus
AUTHORS: abdelmounaim_nouri, Maria L. Rodgers, Daniel L. Bolnick, Natalie C. Steinel

[DESCRIPTION]
This protocol details the Establishment of primary intestinal epithelial cells and leukocytes from the three-spined stickleback, Gasterosteus aculeatus.

[BEFORE_START]

Fish dissection:

Prepare one Petri dish on ice with 10 mL of 1X PBS for collecting the tissue. 

Prepare Two Petri dishes containing 10 mL of PBS 1X, 0.1% povidone-iodine and, two Petri dishes with 10 mL 1X PBS and place inside the biosafety cabinet to sterilize the intestine.


[STEPS]
SECTION: Fish dissection
1. After following approved euthanasia procedures, place the fish’s body on ice.
SECTION: Fish dissection
2. Make a ventral incision from the cloaca to the jaw using sharp surgical scissors.
SECTION: Fish dissection
3.  Make two lateral incisions just behind the opercular flaps down to the lateral line of the fish.
SECTION: Fish dissection
4. Using two pins, secure the fish on its dorsal side on a dissecting pad.
SECTION: Fish dissection
5. To detach the intestine, make a cut at pyloric caeca on one side and the cloaca on the other side. 
SECTION: Fish dissection
6.  Place intestine into a Petri dish containing 10 mL of cold 1X PBS.

SECTION: Fish dissection
7. Using forceps and mini dissecting scissors, open the intestine by making a longitudinal incision.
SECTION: Fish dissection
8. 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 5 min each. 

SECTION: Fish dissection
9. Wash twice for 5 min each in a Petri dish containing 10 mL of cold 1x PBS to remove iodine.

SECTION: Mucus removal:
10. 
Transfer opened gut in 10 mL of mucus removal solution and incubate for 10 min at 17 °C on a gyratory rocker for 10 min.
SECTION: Mucus removal:
11. Resuspend the gut tissue in a fresh 10 mL of mucus removal solution and incubate again incubate for 10 min at 17 °C on a gyratory rocker.

SECTION: Epithelial cells recovery and enzymatic digestion:
12. 
 


Transfer the gut tissue to 10 mL of epithelial cells recovery solution and incubate for 10 min at  17 °C on a gyratory rocker.
SECTION: Epithelial cells recovery and enzymatic digestion:
13. 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 300 x g for 10 min at 17 °C. 
SECTION: Epithelial cells recovery and enzymatic digestion:
16. Collect and save the cell suspension at  17 °C.
SECTION: Epithelial cells recovery and enzymatic digestion:
15. Transfer the intestinal tissue from step 13 to 7 mL of enzymatic digestion solution, then incubate for 30 min at 17 °C on a gyratory shaker.
SECTION: Epithelial cells recovery and enzymatic digestion:
17. Resuspend the remaining intestinal tissue removed from the enzymatic digestion solution in 7 mL of fresh enzymatic digestion solution for a second enzymatic digestion.
SECTION: Epithelial cells recovery and enzymatic digestion:
18. Incubate for an additional 30 min at 17 °C on a gyratory shaker.
SECTION: Epithelial cells recovery and enzymatic digestion:
19. Recover the cell suspension and pool with cell suspensions obtained from step 16 in a 15 ml conical tube and keep at   17 °C.
SECTION: Epithelial cells recovery and enzymatic digestion:
20. Filter the obtained cell suspension through a   mesh cell strainer into a new tube to remove cell clumps.
SECTION: Epithelial cells recovery and enzymatic digestion:
21. Ccntrifuge the obtained unicellular suspension at 300 x g for 10 min at 17 °C.
SECTION: Epithelial cells recovery and enzymatic digestion:
22. Resuspend the cell pellet in 5 mL L15 with 2% FBS and 1% pen Strep.
SECTION: Density gradient:
23. 
Use double-density leukocyte isolation medium to recover all leukocytes from the cell suspension.
SECTION: Density gradient:
24. In a 15 mL conical tube, add 10 mL of the density medium.
SECTION: Density gradient:
25. Carefully layer 5 mL of the cell suspension onto the density medium and mix the two phases.
SECTION: Density gradient:
26. Centrifuge for 20 min at 750 x g at 17 °C.
SECTION: Density gradient:
27. After the density centrifugation, one white layer of cells appears between the L15 medium and Ficoll. Aspirate the top layer of the L15 medium.
SECTION: Cell seeding:
29. Seed the cells into 96 wells plate at a density of 1x106 cells/ml in L15 media with 10% FBS and 1% PenStrep.
SECTION: Epithelial cells recovery and enzymatic digestion:
14. Remove the supernatant and resuspend the epithelial cell pellet in HBSS with 2% FBS and 1% Pen Strep.
SECTION: Density gradient:
28. 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, 300 x g, once with 10 mL of L15 2% FBS and 1% PenStrep.