ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 84

TUNEL assay
TUNEL (terminal deoxynucleotidyl t ransferase mediated d U TP n ick-
e nd l abeling) is a sensitive method to identify apoptotic cells. During
apoptosis, nonrandom DNA fragmentation occurs, and the enzyme TdT
and can be used to attach labeled dUTP to the DNA ends
of the fragmented DNA. The dUTP can be labeled with a variety of
tags, including fluorescent markers or with biotin. The number of cells
with fragmented DNA can be determined using FACS, but this method
works equally well with tissue sections and adherent cells.
Of course, there are non-FACS-based methods for analyzing apop-
tosis. The DNA from cells that are undergoing apoptosis also give a
characteristic laddering due to nonrandom fragmentation, and this can
be viewed on agarose gels (using ethidium bromide stained DNA).
Miscellaneous FACS based assays
Calcium flux
One of the earliest signals in cells when ligand binds the receptor is of-
ten a flux in cellular calcium. FACS can be used to monitor calcium fluxes
upon ligand receptor interactions. Calcium flux is frequently measured
using lndo-1. Because INDO-1 has a different fluorescence emissions
spectra when it is bound to calcium and when it is not, an increase in
cellular calcium will result in a rapid increase in fluorescence intensity.
Cell proliferation
Cell proliferation can be monitored in cells that have been stained with
the dye CFSE (carboxy-fluorescein diacetate, succinimidyl ester). CSFE
contains a fluorescein molecule and diffuses freely into cells. Intracellu-
lar esterases cleave acetate groups on the CSFE which converts it into
a fluorescent, membrane-impermeant dye. The dye remains in the cells
and is not transferred, nor does it (to anyone's knowledge) adversely
affect cellular functions. During each round of cell division, the relative
intensity of the dye is decreased by half, therefore, one can monitor the
number of cell divisions that an individual cell or population of cells is go-
ing through by monitoring the decreased levels of CSFE in the individual
cells up to 3-4 cell divisions.
Gene expression studies
FACS is frequently used to identify cells that have activated the re-
porter gene (for example, in promoter constructs that use GFP, or in