FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 126

centrifugation. Cells were incubated for approximately 30, 60, or 120 min at 37 C and
then centrifuged as before. Supernatants were removed and cells were resuspended in
100 m LAnnexinV staining solution containingAnV-APC. Samples were incubated at
RT in the dark for 15min, then 500 L of HBSS containing mBCl and ASBMS was

added to each tube and vortexed. After 10min of incubation at RT, cells were
immediately analyzed by flow cytometry (Figure 6.1). Triplicate samples were run
for each experiment and an experiment for each compound was run on three separate
days.

m

6.5.1.4 Flow Cytometry The five fluorescent signals (TMRM, 5-DAAF, mBCl,
ASBMS, and Annexin V-APC) were collected as outlined in Table 6.2. A minimum
of 10,000 events was collected per sample. Median fluorescence intensity (MFI)
was recorded for 5-DAAF, TMRM, and mBCl as a measure of lipid peroxidation,
mitochondrial membrane potential, and intracellular glutathione content,
respectively. Apoptotic cells were enumerated by determining the percentage of
events that displayed phosphatidyl serine exposure, yet did not have compromised
plasma membranes as determined by positive staining for AnV-APC and dim/
negative staining for ASBMS (AnV þ /ASBMS ). Cells with compromised
plasma membranes were considered dead/necrotic and were enumerated by
determining the percentage of events that stained brightly for ASBMS (ASBMS þ ).

6.5.2 Results and Discussion

6.5.2.1 Vehicle Effects Vehicle (0.1% DMSO) had a protective effect on relative
intracellular glutathione content that increased in magnitude over time. Measured
mBCl fluorescence increased to 56.41 9.62 of untreated control at 120 min.

No appreciable effect was seen on lipid peroxidation (5-DAAF) or mitochondrial
membrane potential (TMRM). Absolute values of both incidence of apoptosis and cell
death in vehicle-treated samples and trends of increased incidence over time were
similar to those observed in untreated samples.

6.5.2.2 Time Effects All 12 experiments contained untreated control samples. In
order to determine effects of time alone, the average change from the 30min time
point was calculated for the 60 and 120min time points for relative glutathione
content, lipid peroxidation, and mitochondrial membrane potential parameters.
Relative glutathione content measurement was shown to be very sensitive to time
with significant variance from 30 min values at 60 and 120 min. Mean percentage
change in mBCl fluorescence from 30min time point was 40.0 10.1 and

5.1 at 60 min and 120min, respectively. Lipid peroxidation and
mitochondrial membrane potential measurements were less sensitive over time,
but still varied appreciably at 120min and at 60 and 120 min, respectively.
Incidence of apoptosis and cell death both increased slightly over time. Incidence
of apoptosis and cell death of less than 5%was considered inconsequential; therefore,
only the incidence of cell death at 120 min (9.4

73.8

2.3%) was considered a biologically
relevant time-induced stress result. These effects were similar to those seen in cells