ApoptosisReconstitution of activated cytoplasmic extracts wi

Monday, November 24, 2003

Description
Reconstitution of activated cytoplasmic extracts with isolated radioactive nuclei. Analysis of apoptotic activity by quantitative, radioactive DNA fragmentation assays or DAPI staining.

Procedure
Radioactive DNA fragmentation assay

Radioactive nuclei (e.g from ALVA31 cells) were prepared as described in the protocol "Isolation of cell nuclei for the application in a cell-free system". Prior to use in the cell-free system, 5x104 nuclei in NSB (5 l of 107 nuclei per ml) were distributed in 0.5 ml microfuge tubes and were washed once in 50 l DB. The nuclei were then incubated in cytoplasmic extracts (final protein concentration 7.5 mg/ml) in the presence or absence of 10 M cyt c and 1 mM dATP in a total volume of 10 l for 4 h at 37C (650 nuclei/g protein).

Here is an example for a reaction mix for this kind of experiment:

Pipet 5x104 radioactive nuclei in NSB (= 5 l of a stock with 107 nuclei per ml) into 0.5 ml microfuge tubes. Add 50 l of DB. Mix. Spin nuclei down at 800 x g (at 4 degrees celsius) and carefully remove supernatant.
Add x l of DB to the nuclei.
Add 0.3 l of cytochrome c (325 M) and 1 l of dATP (10 mM) to reaction mix.
then y l of cytoplasmic extract.
Incubate for 4h at 37C.
y corresponds to 75 g protein, and x is calculated so that the final volume is 10 l.

After incubation, the nuclei were transferred from the microfuge tubes into the wells of a 96 well plate; the nuclei's DNA was harvested on a glassfiber membrane and the retented radioactivity measured by scintillation counting. Experiments were run in triplicate or pentuplicate for each condition. The percentage of DNA fragmentation was calculated as follows:

( [cpm of nuclei in pure extracts] - [cpm of nuclei in extracts + cyto c/dATP] ) / [cpm of nuclei in pure extracts] x 100

Recipes
Extract Dilution Buffer (DB):

COMPOSITION:
RECIPE for 500 l:

10 mM HEPES (pH 7.0)
5 mM EGTA
50 mM NaCl
2 mM MgCl2
1 mM DTT

supplemented with ATP regeneration system:

2 mM ATP
10 mM phosphocreatine
50 g/ml creatine kinase

100 l of 50 mM HEPES, pH 7.0 incl.
25 mM EGTA
25 l of 1 M KCl
10 l of 100 mM MgCl2
0.5 l of 1 M DTT

-

5 l of 200 mM ATP in water
10 l of 500 mM phosphocreatine in water
10 l of 2.5 mg/ml creatine kinase in KPM buffer

365 l H2O nuclease free

Add DTT and ATP regeneration system always fresh to the buffer, just before use!

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Caspase Assay Buffer (CAB):

COMPOSITION:
RECIPE for 50 ml:

50 mM PIPES
0.1 mM EDTA
10% glycerol
1 mM DTT

838.4 mg PIPES
10 l of 0.5 M EDTA
5 ml glycerol
-

add 40 ml H2O, adjust pH=7.2 using KOH (about 200 l of 1M KOH), then fill up to the final volume of 50 ml.

1mM DTT is always added fresh to the buffer, just before use.

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Lysis Buffer (for DNA isolation from nuclei)

COMPOSITION:
RECIPE for 50 ml:

50 mM Tris-HCl, pH 8.0
10 mM EDTA
0.2 % SDS

5 ml of 0.5 M Tris-HCl, pH 8.2
1 ml of 0.5 M EDTA, pH 8.0
1 ml 10% SDS

add 43 ml H2O

before use, add proteinase K to a concentration of 0.5 mg/ml

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TE Buffer

COMPOSITION:
RECIPE for 50 ml:

50 mM Tris-HCl, pH 8.0
1 mM EDTA

5 ml of 0.5 M Tris-HCl, pH 8.2
100 l of 0.5 M EDTA, pH 8.0

add 44.9 ml H2O

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4% Paraformaldehyde in PBS

RECIPE for 100 ml:

Heat 80 ml H2O to 60C
under stirring add 4g Paraformaldehyde
cover, let stir at 60C, but do not overheat
add 2 drops of 1 N NaOH: solution becomes almost clear, but contains some particles that will not dissolve
add 4 ml of 25x PBS
adjust pH to 7.0 with HCl
bring to final volume with H2O filter solution and store in brown glass bottle at 4C
Solution is good for at least 1 year.

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ApoptosisReconstitution of activated cytoplasmic extracts wi