FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 131

undertaking particularly challenging since so many factors can impact results
including euthanasia (e.g., CO 2 , isofluorane, etc.), blood collection method (e.g.,
blood vessel, needle size, and anticoagulant), blood fraction processed (e.g., lysed
whole blood, mononuclear fraction, and platelet-rich plasma), enumeration method
(e.g., microscopy after centrifugation, microscopy after magnetic enrichment, and
flow cytometry), and primary phenotypic identifier (e.g., cellular morphology alone,
DiI-Ac-LDL uptake, lectin binding, and various cell surface proteins). Lack of
consensus methods also made it difficult to establish a “normal” range for basal
CEC levels since basal levels in humans greatly vary among methods employed [15].
Rat CEC gene expression was also unprecedented although microarray analysis had
been done with magnetically isolated human CEC [16].

6.6.2 Identifying CEC/CEP Markers
Initial experiments in untreated animals used 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethy-
lindocarbocyanine perchlorate-acetylated low-density lipoprotein (DiI-Ac-LDL) that
has been widely used as endothelial identifier. DiI-Ac-LDL has been used by other
investigators in sorting experiments to isolate endothelial cells from explant cul-
tures [17, 18]. This reagent was useful for early method development work but was
logistically impractical for biomarker development and cell sorting with subsequent
gene expression analysis experiments since it requires a 4 h incubation period to allow
uptake. Since DiI-Ac-LDL is ingested through binding to scavenger receptors, it was
postulated that a similar cell population could be identified using an antibody against
said scavenger receptor. CD36 is a type B scavenger receptor and the rat ortholog,
fatty acid translocase (FAT), has been shown to be present on endothelium [19]. Rat
blood samples costained with DiI-Ac-LDL and anti-CD36 exhibited 80% overlap

between labels, therefore anti-CD36 antibody replacedDiI-Ac-LDL in all subsequent
experiments. However, CD36/scavenger receptor is also present on the cell surface of
other cell types in circulation including macrophage/monocytes, erythrocytes, and
platelets. Initially, only monocytic lineage cells were excluded using anti-CD11b/c
since ammonium chloride lysis of RBCs was performed and platelets are easily
excluded based on size. In hindsight, given the sheer number of erythrocytes in blood,
the fact that lysis is not 100% effective, reticulocyte resistance to lysis and very low
number of CECs, it should also have been obvious that erythrocyte lineage would
comprise a large percentage of remaining CD11b/c /CD36 þ cells. Negatively
selecting for erythrocyte lineage was problematic because the only antirat erythroid
antibody available at the time was an IgM isotype. This reagent led to agglutination
and severe clump formation. To circumvent this problem, the cell-permeant DNA
binding dye Hoechst 33342 (HO) was used to identify nucleated cells allowing
negative selection of both erythroid lineage and (free) platelets. Since it was
confirmed that platelets often associated with other cell types, it became necessary
to add anti-CD42d to exclude platelet-associated cells from analysis [20]. Anti-CD3
and anti-CD45R were included to negatively select for T and B lymphocytes,
respectively. A specific reagent to negatively select for NK cells was not necessary
as they are CD11b/c þ in the rat strain (Sprague-Dawley) used. Although some