FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 125

FIGURE 6.1 Multiparameter oxidative stress sample preparation flowchart

culture and an aliquot counted on a hemacytometer to determine cell number and
assess viability using trypan blue. An appropriate volume of cell suspension was
placed in a 50 mL conical tube and centrifuged at 200 g for 5 min at room temperature
(RT). Cells were suspended in a like volume of HBSS and centrifuged again. Cells for
single-color flow cytometry compensation controls were removed, treated, and
stained separately. TMRM (30mL of 500 nM in HBSS) was added to cell pellet
and vortexed followed by incubation for approximately 15 min at 37 C. Cells were
centrifuged as before, resuspended in 30 mL of 5-DAAF (100 nM inHBSS), vortexed,
and then incubated for approximately 60min at 37 C. Sample was removed from
incubator, aliquoted among appropriate number of 12 75 mm polystyrene tubes,

and then centrifuged as before. In some cases, cells were then preincubated with
compound appropriate to alleviate the effect of the toxicant to be tested, which will
be denoted as remedy compounds. Four toxicant/remedy compound pairs were tested:
menadione/NAC, hydrogen peroxide/catalase, FCCP/cyclosporin A, and paclitaxel/
Z-VAD-FMK. Cell pellets were suspended in 500 L of the appropriate remedy

compound or HBSS and incubated for approximately 60 min at 37 C. Treatments
were added at a 2

m

concentration and vortexed before proceeding with no