Gentle lysis of cells in sucrose-containing Mito-Buffer

Monday, November 24, 2003

Description
Lysis in the sucrose-containing Buffer ("Mito-Buffer") is supposed to prevent accidential disrupture of the mitochondria to prevent the leakage of mitochondrial proteins (such as cytochrome c) into the cytosol. Lysis is achieved by using a pestle homogenizer; the number of strokes with the pestle to lyse the cells but leave the mitochondria intact, depends on the cell line. For PC3, DU145, JCA1 and ALVA31 up to 50 strokes appear to be reasonable, whereas mitochondria from LNCaP appear to be more fragile and only up to 30 strokes should be applied. If different compartments are going to be isolated, the cell lysate is centrifuged at different speeds to obtain successively nuclei, mitochondria and cytosol.

Procedure
Harvest cells by trypsinization. Spin cells down at 200xg for 5 min.
Wash cells once with 5 ml of fresh RPMI medium.
Wash cells once with 5 ml icecold PBS.
Resuspend cells in Mitobuffer (approx. 5,000,000 cells in 300 l of buffer).
Lyse the cells by an appropriate number of strokes with the pestle in a glass homogenizer on ice.
Spin lysate at 800 g (3000 rpm in Eppendorf microfuge) for 20 min at 4C
The pellet contains nuclei and intact cells, whereas the supernatant is the cytosol including the mitochondria. If necessary, the supernatant can be spun a second time at 800 g to remove residual nuclei.
The supernatant is then spun at 10,000 g (11,000 rpm) for 20 min at 4C. The pellet contains the mitochondria which should be washed at least once with Mitobuffer and spun down again at 10,000 g. The mitochondria may be lysed by incubation for 15 min in TNC buffer on ice.
The supernatant is centrifuged at 16,000 g (14,000 rpm) for at least 20 min to remove residual mitochodria. 16,000 g seem to be sufficient, but if available, the sample should be spun at 100,000 g for 1h in an ultracentrifuge to remove residual membranes.

Recipes
Mito-Buffer:

COMPOSITION:
RECIPE for 5 ml:

20 mM HEPES (pH 7.5)
1 mM EGTA
1 mM EDTA
10 mM KCl
1.5 mM MgCl2
1 mM DTT
0.1 mM PMSF
2 g/ml Leupeptin
2 g/ml Pepstatin
2 g/ml Aprotinin

2 ml of 50 mM HEPES, pH 7.5 incl.
2.5 mM EGTA and
2.5 mM EDTA
50 l of 1 M KCl
75 l of 100 mM MgCl
5 l of 1 M DTT
50 l of 10 mM PMSF
10 l of 1 mg/ml Leupeptin
10 l of 1 mg/ml Pepstatin
10 l of 1 mg/ml Aprotinin
2.55 ml H2O nuclease free

TNC-Buffer:

COMPOSITION:
RECIPE for 5 ml:

10 mM Tris-acetate (pH 8.0)
0.5% NP-40
5 mM CaCl2
1 mM DTT
0.1 mM PMSF
2 g/ml Leupeptin
2 g/ml Pepstatin
2 g/ml Aprotinin

1 ml of 50 mM Tris-acetate, pH 8.0
25 l of Nonidet P-40 (or IGEPAL, Sigma)
25 l of 1 M CaCl2
5 l of 1 M DTT
50 l of 10 mM PMSF
10 l of 1 mg/ml Leupeptin
10 l of 1 mg/ml Pepstatin
10 l of 1 mg/ml Aprotinin
3.865 ml H2O

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Gentle lysis of cells in sucrose-containing Mito-Buffer