FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 138

FIGURE 6.4 Rat CEC GeneChip statistical analysis revealed dose-responsive genes. Four
representative genes are presented. (a) Colony stimulating factor 2 receptor, b 1 (Csf2rb1). (b)
Voltage-dependent anion channel 1 (Vdac1). (c) Apolipoprotein B (Apob). (d) Endothelium
differentiation sphingolipid G-protein-coupled receptor 1 (Edg1). (a) and (b) increased and
(c) and (d) decreased in a dose-dependent manner relative to mean control expression levels.

to identify viable nucleated cells of erythroid, myeloid, and lymphoid lineages for
comparison with manual cytologic analysis of mouse bone marrow.

6.7.1 Methods

6.7.1.1 Sample Collection Mice were killed by carbon dioxide asphyxiation and
exsanguination. Both femurs were removed and dissected free of soft tissues. Right
femurs were used for flow cytometric analysis and cytospin preparations and left
femurs for bone marrow smear preparation. Previous experiments confirmed no
statistically significant difference in bone marrow differentials among femurs in same
animals.

6.7.1.2 Cytology Bone marrow smears were prepared using standard methods.
Cytospin slides were prepared from collected whole bone marrow and sorted cells.
Cells (2 10 5 ) were washed and resuspended in 250 m L PBS. Cytospins were

prepared using Polysine -coated slides (Erie Scientific Company, Portsmouth,

NH) on a cytospin 2 (Shandon, Inc., Pittsburg, PA) and then fixed and stained
using an Advia S60 automated slide-staining system (Bayer, Tarrytown, NY). Cells
were visualized with a Nikon Eclipse E-800 microscope (Nikon, New York) using
a Plan Apo100X oil objective (Nikon) and photomicrographs were taken with a Spot
RT Slider digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Images
were prepared with Photoshop (Adobe Systems, San Jose, CA).

6.7.1.3 Flow Cytometry Myeloid, lymphoid, and nucleated erythroid
populations were positively identified using fluorochrome-conjugated monoclonal
antibodies. Antibody cocktail consisted of antimouse CD45-APCCy7, ter119-PE,
and CD11b-APC. HO (2 g/ml) were used for nuclear

recognition and dead cell exclusion, respectively. First, 7AAD staining was
performed (10min, RT) and then samples were washed, fixed (1% PFA), and

m g/ml) and 7AAD (1 m