Biology Reference
In-Depth Information
TNF-a, CHX, and propidium iodide (PI ) were from Sigma (St. Louis, MO).
Fluorescein isothiocyanate (FITC)-conjugated annexin-V (ANX-V ) was from
Bender MedSystem ( Vienna, Austria).
Micoplasma-free U937 or its HIV-infected clone U1 was cultured in com-
plete medium ( RPMI-1640 supplemented with 10% heat-inactivated fetal
calf serum (FCS), 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2 mM
l-glutamine) and kept at 37
C in a humidi®ed atmosphere (5% CO
2
in air).
Cells were collected during the log phase of growth, washed in phosphate buf-
fered saline ( PBS) at room temperature, counted, and adjusted to a concen-
tration of 10
6
/ml. Cells were then treated with 4 mM CHX for 2 h, and sub-
sequently with 50 IU/ml TNF-a for 4 or 6 h. At the end of the incubation
period, cells were harvested, washed, counted, and stained as described below.
FLOW CYTOMETRY
The analysis of the samples was performed using a FACScan cytometer ( Becton
Dickinson, San Jose, CA) equipped with an argon-ion laser tuned at 488 nm.
The following parameters were assessed.
Mitochondrial Membrane Potential (
Dc
)
Cells were stained with the lipophilic cationic probe JC-1 (Molecular Probes) as
described (Cossarizza et al., 1993). Staining with this ¯uorescent probe is, in
principle, quite simple. For U1 cells, after incubation with apoptogenic agents,
a cell suspension is adjusted to 0.5±1 10
6
cells/ml and incubated with 2.5 mg/ml
JC-1 in complete medium ( RPMI-1640 with 10% FCS) for 10 min at room
temperature in the dark. During the addition of the dye, or immediately after, it
is important to vortex the tubes, as the solubility of the probe is low in water; in
this manner aggregates of the probe should not be present or should disappear.
Then, the cells are washed twice in PBS, resuspended in PBS (it is also possible
to use PBS with 0.5% paraformaldehyde), and analyzed. It is advisable to pre-
pare a functional ``negative''control, treating a parallel sample with drugs able
to collapse DC, such as the K
ionophore valinomycin (100 nM or more) or the
proton translocator carbonyl cyanide p-(tri¯uoromethoxy) phenylhydrazone
(FCCP, 250 nM). A dramatic change of the ¯uorescence distribution occurs (as
in Fig. 13.3), which indicates where cells are whose mitochondria have a low
Dc. The compensation of FL1 and FL2 either in control or treated samples is
then set.
Changes in
Dc
and Early Apoptosis by Dual Staining with MT and
ANX-V
Thirty minutes before the end of the scheduled incubation period, 200 ml cell
suspension is harvested from each Petri dish, transferred to a suitable test tube,
and the volume is brought to 1 ml with complete medium. These samples are
