Assessing Bad Sub-cellular Localization Under Conditions Associated with Prevention or Promotion of Mitochondrial Permeability Transition-Dependent Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 283

3.1. Preparation
of Samples for
Detection of Bad
by Western Blotting

1. U937 human myeloid leukaemia cells were cultured in
suspension in RPMI 1640 Medium supplemented with 10%
fetal bovine serum, penicillin, and streptomycin at 37° in
T-75 tissue culture flasks gassed with an atmosphere of 95%
air-5% CO 2 . Isolation of cytosolic and mitochondria-enriched
fractions requires at least 5 × 10 6 U937 cells.
2. All the materials required for treatments and cell fractionation
are made ready: inhibitors of 5-LO and PKC, as well as
5-HETE and PGE 2 , at appropriate stock concentrations for
1:1,000 dilution into the cultures; a centrifuge tube for each
sample; a vacuum aspirator; cooled centrifuges, and cold
extraction and lysis buffer.
3. Treatments were performed in pre-warmed saline A containing
2.5 × 10 5 cells/ml. The cell suspension (20 ml) was inoculated
into 50 ml plastic tubes before addition of peroxynitrite.
Peroxynitrite was added on the wall of these tubes and imme-
diately mixed to equilibrate its concentration on the cell
suspension (see Note 9).
4. After treatments (10 min), collect the cells by centrifugation
(3 min, 4°C) at 1,300 × g .
5. Add cold extraction buffer to the cell pellet (35 ml extraction
buffer/1 × 10 6 cells) and incubate for 10 min. Homogenize
the cells by 40 passages through a 26-gauge needle (see
Note 10). Centrifuge the cell lysate (5 min) at 1,000 × g to
remove nuclei, unbroken cells, and cell debris. The resulting
supernatant should then be centrifuged (30 min) at 12,000 × g
to obtain the mitochondrial fraction (pellet). The cytosolic
fraction is obtained from the supernatant upon further purifi-
cation with an additional centrifugation (1 h) at 10,000 × g .
The cytosolic fraction is maintained at 4°C prior to electro-
phoresis. The mitochondrial fraction is suspended in 55 ml
of cold lysis buffer, incubated for 20 min on ice and finally
centrifuged (5 min) at 15,000 × g to obtain the solubilised
enriched mitochondrial fraction.
6. Protein concentration is determined with the Bio-Rad Dye
Binding protein assay (see Note 11).
7. Equal amounts (20 mg, see Note 12) of the mitochondrial
and cytosolic fractions are diluted in loading buffer and incu-
bated for 5-10 min at 95°C.

3.2. SDS-PAGE

1. Carefully clean and extensively rinse the glass plates of the gel
cassette.
2. Prepare a 1.0 mm thick 12.5% gel by mixing 4.3 ml of water,
3.1 ml of 40% acrylamide, 2.5 ml of Lower Tris, 100 ml of
10% SDS, and 14 ml of TEMED. 500 ml of this solution
should be mixed with an excess APS (50-60 µl, promoting a