[Application of carbocyanines fluorescent dye for tracing fetal fibroblast transplant: experiment with rats].
OBJECTIVE
To investigate the feasibility of applying carbocyanines fluorescent dye chloromethyl-benzamidodialkyl carbocyanine (CM-Dil) in tracing fetal fibroblast transplant.


METHODS
Sixteen-day-gestation fetal rat skin tissue samples were harvested and digested with trypsin to isolate fibroblasts. The fibroblasts proliferated in logarithmic phase and were incubated with CM-Dil for 15 minutes. Flow cytometry was used to calculate the proliferate index (PI) so as to determine the effect of CM-Dil on cell proliferation. Labeled fibroblasts were transplanted to the deep partial thickness burn wounds in the back of allogenic rats. Samples were harvested on 0, 1, 3, 7, 14, and 28 days post burns (DPB) and observed under fluorescence microscope in 6 microm section.


RESULTS
The cultured fibroblasts were labeled with CM-Dil within 15 minutes, appearing as red fluorescent discs with bilayer membrane structure. The PI of the labeled fibroblasts was (76.9 +/- 2.8), not significant different from that of the cells not labeled (75.8 +/- 2.0, t = 0.80, P > 0.05). The area with fluorescence markers was small on 1 DPB, began to expand and be distributed in a beaker shape on 3 DPB, and then the bottom of the "beaker" broadened indicating cell proliferation significantly on 7 DPB. Fluorescence signals determined were moderate on 14 DPB, and can be still determined feebly on 28 DPB.


CONCLUSIONS
Proper concentration of CM-Dil shows no cytotoxicity to fetal fibroblasts. CM-Dil is an ideal fluorescent label to fetal fibroblasts for its fairly stable fluorescent tracing and slow quenching in skin tissue within 4 week's duration.
Wang
Yi-bing
YB
Department of Burns & Plastic Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China. wyb0616@yahoo.com.cn
Zhang
Rui
R
Feng
Yong-qiang
YQ
Zhang
Cheng
C
D000818:Animals; D002232:Carbocyanines; D018929:Cell Culture Techniques; D002470:Cell Survival; D017690:Cell Transplantation / Q000379:methods*; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000637:transplantation*; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes; D008297:Male; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D012867:Skin / Q000196:embryology
D002232:Carbocyanines; D005456:Fluorescent Dyes