Biology Reference
In-Depth Information
Fig. 1
(
a
) Illustration of the rat abdominal cavity after complete resection of the gastrointestinal tract and the
liver. Both kidneys (
arrows
) are clearly exposed and can be removed with sterile forceps (
b
)
Flap skin and adhering abdominal wall. Completely remove
the gastrointestinal tract and liver. Remove both kidneys with
sterile forceps. Transfer kidneys to a sterile Petri dish or 50-mL
tube containing PBS.
2. Wash the kidneys with PBS and transfer to a fresh Petri dish
containing a small volume of PBS.
3. Remove capsules and attached blood vessels with pointed for-
ceps (
see
Note 3
).
4. Transfer kidneys to a new Petri dish containing a small volume
of PBS and mince into small pieces (approx 0.1-0.5 mm in
diameter) using a sterile scalpel and/or scissors.
5. Transfer minced kidney pieces (~10 kidneys/25 mL PBS) to a
50-mL sterile conical tube. Add collagenase-dispase solution
to a fi nal concentration of 1 mg/mL.
6. Vortex tissue suspension and incubate for approx 1 h at 37 °C.
Vortex approximately every 15 min for approx 30 s.
7. Wait for 5 min to allow the clumps to settle, and transfer the
supernatant to a new 50-mL sterile centrifuge tube kept on ice.
8. Add new PBS (~25 mL/10 kidneys) containing 1 mg/mL col-
lagenase-dispase to the clumps (in original 50-mL tube), vor-
tex, and incubate for another 1-3 h until the clumps are
dispersed (
see
Note 4
).
9. Pool the supernatant (Subheading
3.1
.,
step 7
) and second
digest and centrifuge for 5 min at 800 ×
g
and 4 °C.
10. Remove the supernatant and suspend the cell pellet in a small
volume of prewarmed (37 °C) DMEM containing 10 % fetal
bovine serum and antibiotics.
11. Disperse the cells with the help of a pipet and dispense the
amount of cells corresponding to one-half kidney into one
