Biomedical Engineering Reference
In-Depth Information
inhibition totally abrogates the process [9]. Z-VAD-FMK was chosen for this study
since it is purported to be a pan-caspase inhibitor, but in hindsight it appears that
a caspase-10-specific inhibitor, such as Z-AEVD-FMK, may better attenuate the
effect of paclitaxel [10]. Z-AEVD-FMK or a combination of Z-AEVD-FMK and
Z-VAD-FMK as well as a higher concentration of inhibitor (100
m
M) is suggested.
6.5.2.4 Assay Design Observations The simultaneous use of ASBMS and mBCl
requires significant fluorescence compensation due to a large amount of spectral
overlap. Proper compensation using appropriate single-color controls is paramount to
achieve accurate fluorescence measurements for mBCl. Multiple titration
experiments were performed to determine working concentrations of TMRM,
5-DAAF, and all tool compounds, as well as time points of interest that were
appropriate for HL-60 cells cultured as described. Changes in culture conditions
of HL-60 cells, the use of a different cell line, and/or the use of primary cells would
likely require similar titrations to optimize experimental conditions to achieve desired
effects from tool compounds. If using to screen compounds of unknown mechanism
of toxicity, a wider range of concentrations may be needed to appreciate toxic effects.
This assay as described for use with HL-60 cells is suitable only for short-term
experiments due to the great stress effects observed over time, even in untreated cells.
In addition, it is conceivable that at least some loss of signal of mBCl, 5-DAAF, and
TMRM at later time points is due to leaching of respective fluorescent reporters from
cells and not due to an actual decrease in intracellular glutathione, increase in lipid
peroxidation, and decrease in mitochondrial membrane potential. Therefore, time
points of 15, 30, 45, and 60min are recommended for future studies with HL-60 cells.
It is recommended that tool compounds, such as those described herein, be included as
positive controls in all experiments applying this assay. Also, due to variation
observed with tool compound treatments, multiple samples should be run
simultaneously in each experiment and then results from multiple experiments
compared to confirm results. This is deemed especially important if investigating
toxicants with unknown modes of action and/or toxicants that may not produce the
magnitude of change seen with positive control tool compounds.
Concentration-dependent changes in relative intracellular glutathione content
(decrease), lipid peroxidation (increase), mitochondrial membrane potential
(decrease), and apoptosis (increase) were induced with the tool compounds mena-
dione, hydrogen peroxide, FCCP, and paclitaxel, respectively, in HL-60 cells. In
addition, secondary effects on other stress parameters, both negative and positive,
were also observed, suggesting cross talk among parameters and/or propagation of the
stress effect to other compartments. This strongly supports the use of multiparameter
analysis during assessment of cytotoxicity, especially if mechanism is a goal of the
experimental design. Effects of time and vehicle (0.1% DMSO) were also observed
and should be considered when performing experiments of this type. Multiparameter
flow cytometry is deemed valid for reporting the effects of known toxicants on
oxidative stress, apoptosis, and cell death parameters simultaneously in HL-60 cells.
This method could be applied as a screening tool to investigate the multistress
potential and mechanism of toxicity of uncharacterized toxicants on HL-60 cells.
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