Biology Reference
In-Depth Information
stained with 50 nM MT for 30 min at 37
C. At the end of the incubation time,
the samples are washed once in PBS and resuspended in 200 ml ANX-V binding
bu¨er (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl
2
), stained
with 5 mL ANX-V, and incubated for 10 more minutes at room temperature
(Salvioli et al., 2000b). The cells are then washed with binding bu¨er to remove
the excess ANX-V, resuspended in PBS (eventually containing paraformalde-
hyde), and analyzed. MT and ANX ¯uorescence are collected in FL2 and FL1
channels, respectively.
Late Apoptosis
The classical appearance of the hypodiploid peak of PI ¯uorescence, according
to Nicoletti et al. ( Nicoletti et al., 1991), is utilized for the quanti®cation of this
parameter, as described ( Barbieri et al., 1992). Brie¯y, cells are resuspended
in hypotonic solution containing 0.1% sodium citrate, 0.1% Triton X-100 and
50 mg/ml PI, and kept for 20 min at 4
C. Then, cells are analyzed on the ¯ow
cytometer, and those with low PI ¯uorescence, i.e., that contain less DNA, and
with the typical change in physical parameters (as revealed by an increase in the
side scatter) are considered apoptotic.
A minimum of 10,000 cells per sample are acquired in list mode and ana-
lyzed with WinMDI 2.8 software (kindly provided by Dr. J. Trotter, Scripps
Institute, La Jolla, CA).
INTERPRETATION OF THE DATA
Many mechanisms can be triggered that provoke the death of immune cells,
including a partial, incomplete activation (i.e., delivery of the ®rst stimulatory
signal, which is not followed by the second), the action of pro-in¯ammatory
cytokines such as TNF-a, and the activation of receptors such as CD95 that
mediate apoptosis, among others (Buttke and Sandstrom, 1994; Collins et al.,
1991; Dive et al., 1992; Falcieri et al., 1993; Green et al., 1992; Grell et al.,
1989; Nagata, 1999; Nagata and Golstein, 1995). However, one of the key
problems that await a solution concerns the individual sensitivity of infected
cells to apoptosis. As a consequence, much less is clear concerning apoptosis in
infected lymphocytes or monocytes, and studies with chronically infected cell
lines are critical to understand the interactions between virus and host that
cause or inhibit cell death. On the one hand, apoptosis is a defense system that
cells activate to destroy agents that are dangerous for the organism. On the
other hand, undesired invaders like viruses gain no advantage in provoking
death of the infected cell, and have thus developed antiapoptotic mechanisms.
Recently, using chronically infected cell lines such as U1 and ACH-2 (derived
from the lymphocytic clone A301), we studied the expression of CD95 (APO-1/
Fas), a member of the TNF receptor family that is deeply involved in this pro-
cess, and its ligand (CD95L) ( Pinti et al., 1999). Flow cytometry revealed that
