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
<
Search WWH ::




Custom Search