CellfreeSystemfortheexaminationofapoptoticactivity

1. Reaction mix
   

   Cytoplasmic extracts were diluted with DB to a concentration of 7.5 μg protein per ml in a total volume of 50 μl. The extracts were activated or not activated by addition of e.g. 10 μM cytochrome c and 1 mM dATP, then 300,000 isolated nuclei in a volume of 1.5 μl NSB (e.g. from Jurkat cells) were added. The reactions were incubated at 37°C for 4 h.

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

   
   

   y corresponds to 375 μg protein, and x is calculated so that the final volume is 50 μl.

   After incubation, the nuclei were either analyzed by DNA laddering or DAPI staining or both, as described below.

   
   

• Pipet x μl of DB into a microfuge tube.

• Add 1.5 μl of cytochrome c (325 μM) and 0.5 μl of dATP (100 mM) to reaction mix.

• Then pipet y μl of cytoplasmic extract into the tubes.

• Add 1 μl of nuclei in NSB (2x10⁸ nuclei / ml).

• Incubate for 4h at 37°C.

2. DAPI staining of nuclei from cell-free system
   

   
   

   
   

• Pipet 10 μl of 4% paraformaldehyde solution (in PBS) onto a glass slide.

• Add 3 μl of the cell-free reaction mix to the fixing solution on slide.

• Cover with glass cover and seal cover-slide border with e.g. nail paint to prevent drying.

• Observe nuclei under fluorescence microscope at 350 nm (UV filter).

• Count at least 150 nuclei and determine apoptotic or normal morphological phenotype.

3. DNA laddering assay with nuclei from cell-free system
   

   
   

• Add 400 μl of Lysis Buffer (containing 0.5 μg/ml proteinase K, freshly added) to the cell-free reaction.

• Incubate over night at 37°C.

• Add 40 μl of 3M NaOAc, pH8.0, mix.

• Add 900 μl of ice-cold 100% ethanol, mix.

• Spin precipitated DNA down at 16,000xg at 4°C for 20 min.

• Dry DNA pellet at the air or in speed vac.

• Add 20 μl of TE buffer containing 0.2 mg/ml RNase A.

• Incubate at 37°C for at least 30 min.

• Add 5 μl sample buffer (5x).

• Load samples onto a 1.5% agarose gel, run at about 4V per cm for about 2h.

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 μl of 10⁷ nuclei per ml) were distributed in 0.5 ml microfuge tubes and were washed once in 50 μl DB. The nuclei were then incubated in cytoplasmic extracts (final protein concentration 7.5 mg/ml) in the presence or absence of 10 μM cyt c and 1 mM dATP in a total volume of 10 μl for 4 h at 37°C (650 nuclei/μg protein).

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

• Pipet 5x10⁴ radioactive nuclei in NSB (= 5 μl of a stock with 10⁷ nuclei per ml) into 0.5 ml microfuge tubes. Add 50 μl of DB. Mix. Spin nuclei down at 800 x g (at 4 degrees celsius) and carefully remove supernatant.

• Add x μl of DB to the nuclei.

• Add 0.3 μl of cytochrome c (325 μM) and 1 μl of dATP (10 mM) to reaction mix.

• then y μl of cytoplasmic extract.

• Incubate for 4h at 37°C.

y corresponds to 75 μg protein, and x is calculated so that the final volume is 10 μl.

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

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

supplemented with ATP regeneration system:

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

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

838.4 mg PIPES
10 μl of 0.5 M EDTA
5 ml glycerol
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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

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

add 44.9 ml H₂O

• Heat 80 ml H₂O to 60°C

• under stirring add 4g Paraformaldehyde

• cover, let stir at 60°C, 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 4°C