Monday, November 24, 2003

Description
Reconstitution of activated cytoplasmic extracts with isolated nuclei. Analysis of apoptotic activity by qualitative DNA laddering assay and DAPI staining.

Procedure
Reaction mix

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

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


Pipet x l of DB into a microfuge tube.
Add 1.5 l of cytochrome c (325 M) and 0.5 l of dATP (100 mM) to reaction mix.
Then pipet y l of cytoplasmic extract into the tubes.
Add 1 l of nuclei in NSB (2x108 nuclei / ml).
Incubate for 4h at 37C.
y corresponds to 375 g protein, and x is calculated so that the final volume is 50 l.

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


DAPI staining of nuclei from cell-free system


Pipet 10 l of 4% paraformaldehyde solution (in PBS) onto a glass slide.
Add 3 l 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.

DNA laddering assay with nuclei from cell-free system


Add 400 l of Lysis Buffer (containing 0.5 g/ml proteinase K, freshly added) to the cell-free reaction.
Incubate over night at 37C.
Add 40 l of 3M NaOAc, pH8.0, mix.
Add 900 l of ice-cold 100% ethanol, mix.
Spin precipitated DNA down at 16,000xg at 4C for 20 min.
Dry DNA pellet at the air or in speed vac.
Add 20 l of TE buffer containing 0.2 mg/ml RNase A.
Incubate at 37C for at least 30 min.
Add 5 l sample buffer (5x).
Load samples onto a 1.5% agarose gel, run at about 4V per cm for about 2h.

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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