Primary Hepatocyte Isolation
Primary hepatocyte isolation
A. Collagen
I coating plate -
6well plate(450ul each), 12well plate(200ul each), dry it and PBS washing. Click to Details
B. media - M199 with 10% FBS + 1% antibiotics.
C. Collagenase D(Roche) Click to Details or Collagenase type 1 or 4 (Worthington) Click to Details
1X EGTA solution and Enzyme solution
1)
Hepatocyte isolation for 1 mice
EGTA Solution : 50ml/mice
Enzyme Solution: 100ml + Collagenase D 50mg (Collagenase Type 1 55mg) + Collagenase P 5mg or Collagenase Type 1 only 65mg------------dissolve by stirring in bottle
2)
Incubate Medium at 42℃
3)
Lift right lobe by swab and
Scoop back of IVC by Mosquito forceps and put on silk behind IVC.
Press upper IVC by swab, you can see IVC become large and easy to
insert. Insert 20G catheter into IVC. Tie
silk by surgeoun’s knot once.
4)
After thoracotomy partly, blood
flow back into catheter, then connect tube and catheter, and cut portal vein immediately.
Thoracotomy completely, then clip IVC
upper side of diaphragm.
5)
Perfuse with EGTA Solution(10ml/min) for 3 min
6)
Perfuse with Enzyme Solution(10ml/min) for 10 min
7)
Tie the silk on IVS as soon as
remove the catheter and cut ligament over the liver. Then remove the liver without unclip!
8)
Put the digested liver on 100mm
petri dish at RT. (m199 NO FBS 20ml<10ml, 10ml>)
9)
And mince well, but gently on 100mm petri dish. (hepatocytes are most
fragile types of cell, you should be careful and handle most gently at every/whole
step --- e.g. pipetting slowly)
10) Filter the digested liver through cell strainer on 50ml Falcon tube.
11) Centrifuge 500rpm for 1
min at 4℃ (x2 to remove other cell clearly)
12) Discard SUP(add 2~3ml m199 10%FBS and suspend, and add 2~3ml m199
/total volume 5ml, count cell)
13) Plate onto Collagen I coated culture flask.
The video version (Hepatocyte isolation) will be uploaded soon
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