Isolation of mitochondria from procyclic T. brucei

1. Harvest the cells by centrifugation. [10min @ 4000 rpm in GS-3 rotor]

Cells have to be in log-phase

2. Wash the cells 2 times in SET (or PBS)

When dealing with serum-containing media, it is better to wash 2 times. You will get better brakeage of the cells in the next step.

3. Resuspend the cells in cold DTE.

The original protocol says to a density of 1.2 x 10⁹ cells/ml, but we rather resuspend to a density of about 500 x 10⁶ cells/ml. Make sure you do not have any clumps, you may achieve this by using a brush or “loosening” the pellet before adding DTE.

Monitor the cells under the microscope. You will notice some swelling of the cells. After about 10 min, you can break the cells.

4. Brake the cells by passing through a 26 gauche needle at 100 lbs/in².

Immediately add 60% sucrose [6 ml / 50 ml lysate].

5. Centrifuge the lysate [15 min @ 9000 rpm in GS-3 rotor]

Aspirate the supernatant, but be carefull: The pellet is very loose.

6. Resuspend the pellet in STM [5ml STM per 30 ml lysate]

add 1/200 of DNAse I and keep on ice for 30 – 60 min.

7. Add STE (equal or more than STM) and spin for 15 min @ 9000 rpm in a GS-3.

Aspirate the supernatant, the pellet should now be well packed (otherwise, the DNAse did not work well).

8a. For renografin gradients:

Resuspend the pellet in 76 % RSTE [4 ml per 100 x 10⁹ cells].

Layer underneath the gradient (20% to 35%).

Centrifuge for 2 h @ 24’000 rpm in a SW27 rotor.

8b. For Percoll gradients:

Resuspend the pellet in 80 % Percoll [4 ml per 100 x 10⁹ cells].

Layer underneath the gradient (20% to 35%).

Centrifuge for 1 h @ 24’000 rpm in a SW27 rotor.

9. Harvest the mitochondria band.

Add an excess of STE and harvest the mitos by centrifugation [15 min @ 11’5000 rpm in a SS-34 rotor]. Wash twice with STE.

Dealing with Percoll is a pain, if you do not resuspend the mitos enough, you will loose them, since Percoll will self-form a gradient in the tube.

Materials

SET: [8.78 g NaCl, 37.22 g EDTA, 1.2 g Tris] per liter, pH 7.9

STE: [85.6 g sucrose, 2.4 g Tris, 0.75 g EDTA] per liter, pH 7.9

STM: [85.6 g sucrose, 2.4 g Tris, 3 ml 1 M MgCl₂] per liter, pH 7.9

DTE: [1 mM Tris, 1 mM EDTA, pH 7.9]

Renografin:

20 % RSTE [26.3 ml 76% Renografin, 8.56 sucrose, 0.24 g Tris, 0.075 g EDTA]

per 100 ml, pH 7.9

35 % RSTE [46.2 ml 76% Renografin, 8.56 sucrose, 0.24 g Tris, 0.075 g EDTA]

per 100 ml, pH 7.9

76 % RSTE [100 ml 76% Renografin, 8.56 sucrose, 0.24 g Tris, 0.075 g EDTA]

per 100 ml, pH 7.9

Percoll:

20 % Percoll [20 ml Percoll, 8.56 sucrose, 0.24 g Tris, 0.075 g EDTA]

per 100 ml, pH 7.9

35 % Percoll [35 ml Percoll, 8.56 sucrose, 0.24 g Tris, 0.075 g EDTA]

per 100 ml, pH 7.9

80 % Percoll [80 ml Percoll, 8.56 sucrose, 0.24 g Tris, 0.075 g EDTA]

per 100 ml, pH 7.9