Biomedical Engineering Reference
In-Depth Information
treatedwith 0.1%DMSO and thereforewere interpreted as time-induced stress effects
with little vehicle effect observed.
6.5.2.3 Data Analysis Due to the nature of mechanism of action of the fluorescent
reporters 5-DAAF, mBCL, and TMRM, a large interexperimental variation was
observed. Therefore, all MFI (median fluorescence) data were normalized as
a percentage change from vehicle control and/or percentage change from
untreated for comparison of trends among like experiments run on separate days.
No normalization of apoptosis and necrosis data was needed since absolute values of
percent apoptotic and necrotic can be compared directly. Percentage change from
vehicle control and untreated may still be computed easily but is of limited utility.
While percentage change analysis may allow a better visualization of trends due to the
fact that small absolute changes may translate to fairly large percentage changes, this
may also overemphasize a change that is not biologically relevant. For example, an
apoptotic index of 2% in treated versus 1% in control is a 100 percent change from
control (increase), but reporting this as such misleadingly implies great biological
significance. Therefore, it is suggested that only absolute percentages are analyzed
and presented for apoptosis and cell death parameters.
Test Pair 1: Menadione and NAC Menadione consistently produced a concentration-
and time-dependent decrease in relative intracellular glutathione level, the primary
target parameter for this tool compound. Mean percentage change in mBCl
fluorescence from control was
84.1
3.5,
89.3
2.9, and
97.7
5.0 at 30,
60 and 120min, respectively,
M menadione. Lipid
peroxidation was evident at 120min with all concentrations, but inconsistent
across experiments. Decreased mitochondrial membrane potential was observed at
30min with 100
in cells treated with 100
m
m
M; at 60min with 30 and 100
m
M; and at 120min with 10, 30, and
100
M. A
slight increase inmitochondrial membrane potential over untreated control was seen at
60min with 10
m
M. The magnitude of the decrease at 120min was similar for 10 and 30
m
M similar in magnitude to that seen in the vehicle control. There
was no effect on apoptosis. An appreciable increase in cell death was observed
with 100
m
M menadione on
relative glutathione content. NAC-treated cells lost only 12.7% of mBCl fluorescence
at 120min compared to 97.7% in cells that did not receive NAC. NAC also greatly
reduced the amount of menadione-induced cell death and completely abrogated effects
on lipid peroxidation and mitochondrial membrane potential. In fact, measured
TMRM and 5-DAAF fluorescences were at levels above untreated.
m
M at 120min. NAC greatly abrogated the effects of 100
m
Test Pair 2: FCCP and Cyclosporin A FCCP consistently produced a concentration-
dependent decrease in mitochondrial membrane potential, the primary target for this
tool compound. This effect was observed at all time points, but the best separation in
effect was at 30min due to the fact that mitochondrial membrane potential decreased
over time in the absence of compound treatment. FCCP effects on relative
intracellular glutathione content varied depending on concentration and time, but
in all cases values were above untreated controls at the corresponding time point. The
Search WWH ::
Custom Search