Jw. Ford et al., PKH26 AND I-125 PKH95 - CHARACTERIZATION AND EFFICACY AS LABELS FOR IN-VITRO AND IN-VIVO ENDOTHELIAL-CELL LOCALIZATION AND TRACKING, The Journal of surgical research, 62(1), 1996, pp. 23-28
PKH26, a fluorescent cell label, and PKH95, a I-125-radioactive cell l
abel, are both potentially valuable endothelial cell labels because th
ey bind irreversibly within cell membranes. These labels would be part
icularly well suited to tract transplanted endothelial cells in vivo.
However, no previous studies documenting lack of transfer of the label
to unlabeled endothelial cells, as well as the effect of the label on
endothelial cell function, have been undertaken. The purpose of this
study was to determine the optimal method of endothelial cell (EC) lab
eling with PKH26 and PKH95, whether significant EC-to-EC transfer of t
he label occurs, the effects of these labels on EC proliferation and m
embrane function, and the feasibility of using these labels for long-t
erm quantitative EC tracking in vivo. Canine ECs in confluent monolaye
rs or in cell suspension were labeled by exposure to 1.0 or 5.0 mu M P
KH26 for 1, 3, or 5 min. Cell viability was determined by trypan blue
exclusion. The percentage of cells labeled and their fluorescence inte
nsity were determined in a fluorescent-activated cell sorter (FACS). E
ffect of the label on cell function was assessed by measuring EC proli
feration rates as well as intercellular adhesion molecule (ICAM) expre
ssion before and after induction with tumor necrosis factor (TNF). To
determine if transfer of the label occurs, both labeled and nonlabeled
ECs were grown in coculture and subjected to FAGS analysis. For in vi
vo cell tracking, doubly labeled ECs were injected into the femoral ar
tery of rat hindlimbs, and whole-body tissue analysis was undertaken t
o determine labeled-EC distribution at 60 days. Endothelial cells were
labeled with equal efficiency as monolayers or in suspension. Labelin
g had no effect on EC proliferation rates nor on TNF-induced upregulat
ion of ICAM expression. Coculture experiments revealed no significant
label transfer to nonlabeled ECs. In vivo cell tracking studies docume
nted that 8% of label remained within the skeletal muscle capillaries
at 60 days after injection. PKH26 and PKH95 labels incorporate stably
into EC membranes, do not alter endothelial cell function, and provide
a precise means for quantitative EC tracking and histologic localizat
ion both in vitro and in vivo. These labels should prove to be very us
eful for studies of endothelial cell biology and transplantation. (C)
1996 Academic Press, Inc.