Cell-free System for the examination of apoptotic activity
in cytoplasmic extracts

Introduction
 In our lab we use the term 'cell-free system' when we talk about the examination of apoptotic activity in cytoplasmic extracts. The cytoplasmic extracts may be prepared from cells which were treated in culture with an apoptosis-inducing agent or from untreated cells. In the latter case, apoptotic activity can be induced by addition of apoptotic stimuli (such as recombinant active caspases or cytochrome c) to the extracts. Apoptotic activity in the cytoplasmic extracts can be examined by the measurement of enzymatic caspase-acticity, by western blots of proteins processed during apoptosis (caspases and their substrates), or by using isolated cell nuclei as indicators for apoptotic factors inducing nuclear morphological changes and/or DNA fragmentation. It was in 1993 when the first paper described that a cell-free system can mimic characteristic features of apoptosis in intact cells (Lazebnik et al., 1993, J. Cell Biol., 123(1): 7-22). Indeed, it has become clear that the apoptotic pathways acting in the cytoplasm function independently from the nucleus and thus, cell-free systems appear to be appropriate model systems which represent at least part of the apoptotic machinery and signaling mechanisms. Cell-free systems have been previously successfully applied in the dissection of biochemical mechanisms during the apoptotic process, such as the identification and characterization of the 'apoptosome', AIF, and the DNA fragmentation factor ICAD (Zou et al., 1997, Cell, 90:405-413; Susin et al., 1999, Nature, 397: 441-446; Enari et al., 1998, Nature, 391: 43-50). Also signaling pathways such as the caspase-cascade have been studied under cell-free conditions, poviding insight into activation patterns and inhibitor-specificities (Mesner et al., 1999, JBC, 274 22635-22645; Faleiro et al., 1997, EMBO, 16(9): 2271-2281; Takahashi et al., 1997, Oncogene, 14: 2741-2752). The protocols described below are derived essentially from Lazebnik et al., 1993, J. Cell Biol., 123(1): 7-22 and Fearnhead et al., 1997, Genes and Dev., 11: 126-1276.

Activation of apoptotic activity in cytoplasmic extracts and measurement of the resulting caspase enzymatic acticity
  1. Activation reaction

    Cytoplasmic extracts were activated according to following protocol (as an example, cytochrome c/dATP are used as apoptotic stimuli). The components were mixed in the given order and then incubated at 37°C usually for 45 min.

    x µl Dilution Buffer (DB) containing ATP regeneration system +
    1.5 µl cytochrome c from bovine heart (50 µM) +
    1.5 µl dATP (10 mM)+
    y µl cytoplasmic extract (37.5 µg protein)

    x was so adjusted that the total volume was 15 µl and thus the final protein concentration at 2.5 µg/µl.

    When active recombinant caspases (such as caspase-8) are used for activation of the extracts, 100 ng of recombinant caspase is added instead of cytoc/dATP.

    In case that the effect of inhibitors was studied, such as Ac-DEVD-fmk, or zVAD-fmk, the inhibitor at concentrations ranging from 100 nM up to 100 µM was preincubated together with the extract in DB for 5 min, then cyto c/dATP was added.

  2. Caspase enzymatic assay

    After incubation of the extracts in the presence or absence of activating stimuli (cytochrome c/dATP or active recombinant caspase), caspase enzymatic activity was measured according to following protocol:

    200 µl of Caspase Assay Buffer (CAB) containing 20 µM fluorescent substrate (Ac-DEVD-amc Ac-YVAD-amc, Ac-VEID-amc; Calbiochem) was added to the activation reaction. The samples were transfered into the wells of a 96 flat-bottom well plate. After incubation in the dark for 30 min at RT, fluorescence was measured at 360/460 nm with a FL500 fluorimeter (Bio-Tek). If a standard-curve of various concentrations (0 up to 5 µM in CAB) of aminomethylcoumarin (amc) is measured, the enzyme activity can be calculated and expressed as pikomoles of substrate (amc) hydrolyzed per microgram of protein (in the extract) per minute [pmol • µg-1 • min-1] or just as change of the amc concentration in nanomolar per microgam of protein [nM • µg-1].

Activation of apoptotic activity in cytoplasmic extracts for western blot analysis
  1. Activation reaction

    Cytoplasmic extracts were activated according to following protocol (as an example, cytochrome c/dATP are used as apoptotic stimuli). The components were mixed in the given order and then incubated at 37°C usually for 1 h.

    x µl Dilution Buffer (DB) containing ATP regeneration system +
    0.46 µl cytochrome c from bovine heart (325 µM) +
    0.3 µl dATP (100 mM)+
    y µl cytoplasmic extract (75 µg protein)

    x was so adjusted that the total volume was 30 µl and thus the final protein concentration at 2.5 µg/µl.

    When active recombinant caspases (such as caspase-8) are used for activation of the extracts, 100 ng of recombinant caspase is added instead of cytoc/dATP.

    In case that the effect of inhibitors was studied, such as Ac-DEVD-fmk, or zVAD-fmk, the inhibitor at concentrations ranging from 100 nM up to 100 µM was preincubated together with the extract in DB for 5 min, then cyto c/dATP was added.

  2. Western Blot Analysis

    After incubation of the extracts in the presence or absence of activating stimuli (cytochrome c/dATP or active recombinant caspase), western blots were performed loading usually 20 µg protein in sample buffer per lane on a 4-20% SDS-PAGE gel.

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

  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:

    • 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.

    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.

  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.

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

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

MATERIAL

Extract Dilution Buffer (DB):

COMPOSITION:

RECIPE for 500 µl:

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 µg/ml creatine kinase

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

-

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

365 µl H2O nuclease free

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

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 µl of 0.5 M EDTA
5 ml glycerol
-

add 40 ml H2O, adjust pH=7.2 using KOH (about 200 µl 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.

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

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 µl of 0.5 M EDTA, pH 8.0

add 44.9 ml H2O

4% Paraformaldehyde in PBS

RECIPE for 100 ml:

  • 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

Solution is good for at least 1 year.
Work under a hood!!!