Biology Reference
In-Depth Information
Digitonin
F
i
Triton X-100
F
d
F
b
0
100
200
300
400
Time (s)
Fig. 5
Procedure for assessing dye compartmentalization. REF52 fibroblast incubated with 1
m
M
Fura-2 AM dispersed with Pluronic F-127 in Minimal Essential Medium (MEM) for 60 min in an air
incubator at 37
C.Measurement was done inHanks' Balanced Salt Solution (HBSS) containing 2.6 mM
EGTA (su
Y
cient to reduce extracellular [Ca
2
þ
]to
<
1
m
M), and bu
V
ered at pH 7.4 with HEPES.
The concentration of digitonin was 20
m
M; that of Triton X-100, 1%, w/v. (a.u. = arbitrary units).
and release cytosolic dye, whereas 1% Triton X-100 can permeabilize and release
dye from subcellular organelles (e.g., see
Kao et al., 1989
). The procedure consists
of monitoring total fluorescence from a cell or a field of cells, preferably bathed
in low-Ca
2
þ
medium,
10
and treating the cells first with digitonin and then
with Triton X-100.
Figure 5
shows the procedure being applied to a cell loaded
with Fura-2 AM. The fluorescence measured before treatment (F
i
) represents
contributions from cytosolic and compartmentalized dye plus background.
After digitonin release of cytosolic dye, the fluorescence measured (F
d
) represents
compartmentalized dye plus background. The fluorescence level attained after
Triton X-100 treatment is considered the background (F
b
). The fraction of total
intracellular dye that is compartmentalized in organelles is simply (F
d
F
b
)/
(F
i
F
b
).
Nominally Ca
2
þ
-free medium (i.e., medium from which Ca
2
þ
salts have been omitted) or, better
still, medium containing su
10
cient EGTA to make [Ca
2
þ
]
1
m
M (see
Section IV.B.1
). It is important to
keep [Ca
2
þ
] low in this experiment because when [Ca
2
þ
] is elevated, permeabilized cells lose their
integrity rapidly, and dye molecules may leak from organelles even before Triton is applied.
Y
<