PMID- 10452862
OWN - NLM
STAT- MEDLINE
DCOM- 19990923
LR  - 20141120
IS  - 0022-4804 (Print)
IS  - 0022-4804 (Linking)
VI  - 86
IP  - 1
DP  - 1999 Sep
TI  - Optimizing fluorescent labeling of endothelial cells for tracking during 
      long-term studies of autologous transplantation.
PG  - 9-16
AB  - BACKGROUND: The fluorescent marker PKH26 has been demonstrated to be useful for 
      the tracking of endothelial cells in short-term studies; however, the optimal 
      labeling conditions for long-term implants have not been determined. This study 
      was designed to evaluate the effects of PKH26 on endothelial cell proliferation 
      and to identify labeling conditions that would yield the greatest fluorescence 
      over time without adversely affecting cell viability. MATERIALS AND METHODS: 
      Canine jugular vein endothelial cells (CJVECs) were labeled with 0. 04 microM 
      PKH26. Proliferation of labeled and control cells was assessed for 8 consecutive 
      days by [(3)H]thymidine uptake. In a second experiment, CJVECs were labeled at 
      concentrations of 0, 5, 8, 10, and 20 micromol/L. Cells were maintained in 
      culture for 60 days. The fluorescence intensity of each cell population was 
      measured using two techniques. At baseline and at 60 days, fluorescence was 
      measured using a fluorescence-activated cell sorter. On days 14, 28, 45, and 60 
      fluorescence was measured by constructing gray-scale histograms from 
      photomicrographs taken of each flask under rhodamine illumination. Mean viable 
      cell number for each concentration was determined after 60 days. RESULTS: In the 
      first experiment, PKH26-labeled and unlabeled CJVECs demonstrated nearly 
      identical growth curves, suggesting that PKH26 had no adverse effect on 
      proliferation. In the second experiment, after 60 days, the 10 and 20 microM 
      groups displayed greater fluorescence by histogram than the 0, 5, or 8 microM 
      groups; however, they were not significantly different from each other (mean 
      intensity 8.2 vs 9.1, P > 0.05, Student-Newman-Keuls test for multiple 
      comparisons). Over 60 days, the cells labeled with 20 microM PKH26 experienced 
      the only significant decrease in viable cells compared to the unlabeled group 
      (5.5 x 10(5) vs 9.6 x 10(5) cells/flask, P < 0.05). Importantly, we observed no 
      significant differences in cell number between the 10 microM group and the lower 
      concentrations compared to the unlabeled cells (P > 0.05). CONCLUSIONS: We 
      conclude that a concentration of 10 microM PKH26 provides the optimal labeling 
      condition for endothelial cells when long-term tracking is desired.
CI  - Copyright 1999 Academic Press.
FAU - Fox, D
AU  - Fox D
AD  - Department of Surgery, Loyola University Medical Center, Maywood, Illinois 60153, 
      USA.
FAU - Kouris, G J
AU  - Kouris GJ
FAU - Blumofe, K A
AU  - Blumofe KA
FAU - Heilizer, T J
AU  - Heilizer TJ
FAU - Husak, V
AU  - Husak V
FAU - Greisler, H P
AU  - Greisler HP
LA  - eng
GR  - R01 HL41272/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Surg Res
JT  - The Journal of surgical research
JID - 0376340
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Organic Chemicals)
RN  - 0 (PKH 26)
SB  - IM
MH  - Animals
MH  - Cell Count/drug effects
MH  - Cell Division/drug effects
MH  - Cell Survival/drug effects
MH  - Cell Transplantation
MH  - Dogs
MH  - Dose-Response Relationship, Drug
MH  - Endothelium, Vascular/*cytology
MH  - *Fluorescent Dyes/pharmacology
MH  - In Vitro Techniques
MH  - *Organic Chemicals
MH  - Osmolar Concentration
MH  - Time Factors
MH  - Transplantation, Autologous
EDAT- 1999/08/24 00:00
MHDA- 1999/08/24 00:01
CRDT- 1999/08/24 00:00
PHST- 1999/08/24 00:00 [pubmed]
PHST- 1999/08/24 00:01 [medline]
PHST- 1999/08/24 00:00 [entrez]
AID - S0022-4804(99)95597-X [pii]
AID - 10.1006/jsre.1999.5597 [doi]
PST - ppublish
SO  - J Surg Res. 1999 Sep;86(1):9-16. doi: 10.1006/jsre.1999.5597.