(1) Cell culture. It is very important to have healthy log phase cells to start the culture. Do daily subcultures until they are growing well.
Day 1 - Dilute healthy log phase cells 1:1 with fresh medium
Day 2 - 9AM - Inoculate cultures with cells at 0.6 x 106 cells/ml.
Day 4 - 9 AM - Harvest cells. Cell concentration should be around 150 x 106 cells/ml (late log).
Cell counts - hemocytometer or Coulter counter
Volume of DTE to use = Total number of cells/ 1.2 109 ml.
Harvest cells in Sorvall GS3 rotor - 10 min at 5000 rpm.
Wash cells with cold SET. Pellet cells in GS3 rotor 10 min at 5000 rpm.
Resuspend cells without clumps in cold DTE. Can use brush to resuspend
pellets or by shaking or pipetting.
Monitor swelling of cells in phase microscope. Should be able to see the swollen
kinetoplast-mitochondrion with the kDNA at the periphery next to the flagellum
basal body. Add more DTE if necessary to get swelling of cells. When cells are
beginning to burst but the kinetoplast-mitochondrion is still intact, pass
lysate through #26 needle at 100 psi. We use a Millipore 600 ml polycarbonate
container or a Millipore 5 l Pressure device (Cat # XX6700P05) depending on the
volume. Pressure is applied with compressed air. A leurlock for the syringe
needle is attached to the exit and a quick disconnect to the inlet. If clogging
occurs, close valve and replace needle.
- monitor breakage by observing lysate after passage through needle in phase
microscope. If you still have intact cells at this point, the final preparation
will be contaminated with cells. Rapidly add sucrose to 0.25 M from 60% stock (6
ml/ 50 ml lysate) to shrink the mitochondrion to a refractile body.
Pellet mitochondria. SS34 rotor 10 min at 11,500 rpm or GS3 rotor for 15 min at 9000 rpm, or GSA rotor for 15 min at 11,000 rpm. The pellets will be loose due to the presence of DNA. Aspirate supernate carefully.
Resuspend pellets in STM - 5 ml per 30 ml lysate. Add 3 mM MgCl2 (1/133 dilution of 1 M stock). Add DNase I (1/200 of stock). Keep on ice for 30 m in to 1 hr.
Add equal volume STE and pellet mitochondria as above. Pellets should be well packed. If not, the DNAse did not digest all the DNA.
Resuspend pellets in 76% Renografin (4 ml 76% RSE per 1 l original culture). Vortex and let sit 5 min to remove bubbles. Layer 4-5 ml per tube underneath gradients (32 ml, polyallomer tubes) with a 12 ml syringe using polyethylene tubing. The lysate should remain under the gradient. If not, then add more 76% Renografin. Centrifuge 2 hr at 24,000 rpm in SW28 rotor. You should be able to see a discrete band just above the 76% Renografin containing the mitochondria. The position of the band may vary depending on the concentrations of your upper and lower Renografin solutions. Tap gradients by side puncture with #18 needle.
Dilute with STE (2 vol). Pellet mitochondria in SS34 for 15 min at 11500 rpm, or in GSA for 10 min at 11,000 rpm. Wash once in STE. Depending on what you want to do, you can either freeze the pellets at -80oC or resuspend in K-buffer at 0.5 mg/ml and freeze in aliquots.
SOLUTIONS
1. DTE – Prepared fresh
1/200 dilution of 0.2 M Tris-HC1 (pH 7.9 at 5° C).
1/200 dilution of 0.2 M EDTA (pH 7.9).
2. STE (or STM)- 85.575 g sucrose
100 ml 0.2 M Tris-HC1 (pH 7.9)
10 ml. 0.2 M. EDTA or 2 ml 1.0 M MgCl2
Bring volume to 1 liter.
Store frozen.
3. 76% RSTE – 8.556 g sucrose
(From Bracco Diagnostics- RenoCal 76 - Cat No. 086032. This contains 660 mg diatrioate meglumine and 100 mg diatrizoate sodium and 0.1 mg calcium dosodium EDTA as a sequestering agent per ml. pH is adjusted to 6.0-7.7 with sodium carbonate and NaOH or HCl. Each ml contains 3.69 mp sodium and 370 mg organically bound iodine. The viscosity is low. The solution is hypertonic to blood with an osmolarity of 1870 mOsm/kg. It is stored under nitrogen.)
50 m l 0.2M. EDTA
100 ml with stock 76% Renografin
Store frozen and light-free.
4. 20% RSTE – 26.3 ml 76% Renografin
8.56 g sucrose
10 ml 0.2 M Tris HC1 pH 7.9
50 m l 0.2 M EDTA
Bring volume to 100 ml.
Adjust to 1.14 – 1.15 g/ml by adjusting refractive index to 1.3720.
5. 35% RSTE – 46.2 ml 76% Renografin
8.55 g sucrose
10 ml 0.2 M Tris HC1 pH 7.9
50 m l 0.2 M EDTA
Bring volume to 100 ml.
Adjust to 1.26 g/ml by adjusting refractive index to 1.4020.
The gradients can be made in advance by first loading the lower solution
and freezing. Then layer the upper solution and refreeze. Thaw overnight
before using and the gradient is created by the freeze-thaw.
6. DNase I (RNase-free) 2 mg/ml in 10 mM-Tris HC1 (pH 7.4),10 mM
CaC1, 50% glycerol.
7. 0.2 M Tris-HC1 – 12.114 g/500 ml.
Titrate with HCl at 5 C to pH 7.9.
8. 0.2 M EDTA – 37.225 g/500 ml
Titrate to pH 7.9 with NaOH.
9. SET:
0.15 M NaCl
0.1M EDTA (pH 8.0)
10 mM Tris (pH 8.0)
10. 60% sucrose
11. K-buffer:
20 mM HEPES (pH 7.5)
10 or 20% glycerol, 20 or 100 mM KCl
0.2 mM EDTA