Hepatocyte (Hc) and non-parenchymal cells (NPC) isolation protocol

From: Juli Bai, Feng Liu (UTHSA), Nov 2019

1. Anesthetize the mice, using Ketamine/Xylazine. (Ketamine/Xylazine conc. used was 80mg/Kg & 10mg/Kg respectively.)

2. Prepare the animal for surgery wiping the abdomen with ethanol, spread the limbs, and secure them to the dissection board.

3. Dissect the peritoneal cavity and push the intestines to the right side using a cotton-tip; identify both the inferior vena cava and hepatic portal vein.

4. Make the liver perfusion throughout inferior vena cava. Insert the needle through the vena cava and hold the needle in place with a clamp in order to secure it.

5. Cut the hepatic portal vein.

6. Pass 30 mL EGTA buffer (pH = 7.4) (37°C preheated) slowly.

7. Pass 6 – 9 mL/mouse Collagenase type 2 (0.025g in 50 mL, 37°C preheated). Collagenase II final conc.: 0.5 mg/mL.

8. When the liver is sufficiently digested (be soft), cut off the liver (make sure the liver is intact at this point) and transfer it to 1X HBSS/HEPES (with Ca2+ and Mg2+) buffer in a Petri dish while working on ice.

9. Remove the liver capsule by cutting the liver lobes and continue until all of the big clumps are gone.

10. Filter through 100-µm strainer.

11. Spin 50g x 2 min at 4°C.

12. Separate the supernatant (NPC) and Pellet (HC). Collect the supernatant (NPC) into a coated 50 mL tube (Tubes were coated with BSA to reduce sticking of Kupffer Cells to the tube), put NPC on ice & re-suspend the pellet (HC) with 1X PBS + 2% FBS.

13. Wash the HC with 1X PBS + 2% FBS twice by repeated centrifugation and re-suspension (50g x 2 min at 4°C),

14. Similarly, collect all supernatants which contain NPC from same mouse in tube and then centrifuge at 50g for 2min to discard the pellet (the rest of hepatocyte), and then centrifuge at 600g for 10min to get NPC pellet,

15. Re-suspend NPC in 1X RBC lysis buffer, put on ice for 6-10 min, dilute with 1X PBS.

16. Centrifuge at 600 g x 5 min, resuspend in 1X PBS with 2% FBS.

17. If the viability is low, Percoll density separation step can be used to remove dead cells or cell debris.