0.25 M sucrose in 10 mM HEPES buffer, pH 7.5 (Homogenization buffer)
0.25 M sucrose in 10 mM HEPES, pH 7.5 + 1 mM EDTA (Suspension buffer)
Janus Green B
Hemacytometer and microscope
Sacrifice and exsanquinate a rat that has not been fed for at
least 24 hours prior to lab.
Remove the liver and weigh it.
Add the liver to a beaker, and for each gram of liver, add 9.0
ml of 0.25 M sucrose in 10 mM HEPES buffer, pH 7.5. This will
produce a 10% brei, a term used to indicate a homogenized
Add the brei to centrifuge tubes and centrifuge at 4,500 xg
for 10 minutes at 4° C.
Decant the supernatant into clean centrifuge tubes and discard
Recentrifuge the supernatant at 16,000 xg for 25 minutes at 4° C.
Decant and discard the supernatant. Resuspend the pelleted
mitochondria in 20 ml of 0.25 M sucrose in 10 mM HEPES. Skip to
Optional: if cleaner mitochondria are desired,
resuspend in 20 ml of 0.25 M sucrose in 10 mM HEPES + 1 mM EDTA and
perform steps 8 and 9.
Recentrifuge the suspended pellet at 16,000 xg for 25 minutes
at 4° C.
Decant and discard the supernatant. Resuspend the washed
pellet in 20 ml of fresh sucrose without EDTA and place the
suspension in an ice bath until further use is required. The
suspension will remain active for approximately 4-6 hours if kept
Mix a few drops of Janus Green B solution with 0.1 ml of
mitochondrial suspension. Place one drop of this mixture in a
hemocytometer and determine the number of mitochondria per ml. If
there are too many mitochondria to count, make serial dilutions
of 1/10 to 1/1000 and recount. Diluted mitochondria must be
counted rapidly. They are not stable and will decompose if not
counted within a few minutes of the dilution.
Record the number of mitochondria/ml______________