FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 124

TABLE 6.1 Multiparameter Oxidative Stress Tool Compounds

Physiological
End Point

Concentrations
( m M)

Remedy
(conc.)

Toxicant Vehicle

Menadione Glutathione 0, 10, 30, 100 0.1% DMSO NAC (1mM)

FCCP Mitochondrial

membrane
potential

0, 0.1, 0.3, 1 0.1% DMSO Cyclosporin

A (20 m M)

Paclitaxel Apoptosis 0, 10, 30, 100 0.1% DMSO Z-VAD-FMK

(25 m M)

Hydrogen
peroxide

Lipid
peroxidation

0, 10, 30, 100 Water Catalase

(100 units)

10 6 cells/mL in T150 culture flasks at
37 C in a 5% CO 2 humidified atmosphere. Cells were harvested the day before a
planned experiment and replated at a density of 5

and 100 m g/ml streptomycin at a density of 1

10 5 cells/mL.

6.5.1.2 Test Articles and Reporters Table 6.1 defines the model toxicants and
remedies evaluated (end point, vehicle, concentrations, and known remedy
conditions). Table 6.2 provides information on the fluorescent reporters used (end
point, interpretation, concentration, and excitation, and filter collection wavelength
information).

6.5.1.3 Experimental Design Figure 6.1 outlines in a flowchart format the

10 7 HL-60 cells were harvested from following procedure. Approximately, 3

TABLE 6.2 Multiparameter Oxidative Stress Fluorescent Reporters

Laser
Excitation
(nm)

Fluorescence
Band-Pass
Filter (nm)

Fluorescent
Reporter

Physiological
End Point Interpretation Concentration

5-DAAF Lipid

peroxidation

# MFI ¼
" lipid
peroxidation

100 nM 488 530/30

TMRM Mitochondrial

membrane
potential

# MFI ¼
# potential

500 nM 488 575/26

mBCl Intracellular

glutathione

# MFI ¼
# glutathione

40 m M 350 485/22

ASBMS Loss of plasma

membrane
integrity

% Positive ¼
% cell
death

10 m M 350 530/30

% Positive ¼ %
apoptotic

AnnexinV-
APC

Phosphatidyl
serine
exposure

Per vendor s
instructions

633 660/20