ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 83

the investigator can instruct the FACS to collect individual populations
of cells and put them in individual tubes or even put single cells with the
desired phenotype in individual wells of a 96-well microtiter plate. This
allows for rapid cloning of cells as well as for study of subpopulations
under defined in vitro conditions. FACS can also be used to isolate and
study distinct subtypes of cells in certain tumors, etc.
It should be noted that while most FACS staining is performed on intact
cells, intracellular proteins can also be evaluated using FACS by first
fixing and permeabilizing the cells. Common agents for permeabilization
are discussed in Chapter 5.
In addition to analyzing and isolating cells based on cell surface char-
acteristics, FACS can be used for studying a number of other cell char-
acteristics. Some of these are briefly outlined below.
DNA content and cell cycle analysis
Propidium iodide (PI), DAPI
and Hoechst dyes bind to DNA and become fluorescent.
Because these dyes bind DNA stoichiometrically, they can be used to
analyze DNA content and also provide cell cycle information. DNA stain-
ing can be used to study cell cycle since relative DNA content can show
the proportion of cells in G1, G2 and S phases of the cell cycle. Nei-
ther PI nor DAPI enter live cells, but some of the Hoechst dyes do, and
therefore the analysis is generally performed after cell fixation or perme-
abilization. In addition, cells undergoing apoptosis can be discriminated
from live cells based on their forward and side scatter characteristics as
well as DNA characteristics.
Apoptosis
Apoptosis, or programmed cell death, is one of two major types of cell
death. Apoptosis can be distinguished from necrotic death by a number
of characteristic properties of the apoptotic cells. The FACS can be used
to identify the cells in several different ways.
Annexin V
Annexin V is a member of a calcium and phospholipid binding family of
proteins that binds to phosphatidyl serine. Phosphatidyl serine is usually
on the inner leaflet of the plasma membrane but is exposed on the
surface of apoptotic cells. When the Annexin V is fluoresceinated and
used in combination with PI, the apoptotic cells can be discriminated on
the basis of low PI staining and high Annexin V fluorescence.