Optimization of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester for labeling human intervertebral disc cells in vitro.
We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 microM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 microM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultrastructural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.
Gruber
H E
HE
Orthopaedic Research Biology, Carolinas Medical Center, Charlotte, North Carolina, 28232, USA. hgruber@carolinas.org
Leslie
K P
KP
Ingram
J A
JA
Hanley
E N
EN
D002470:Cell Survival; D002478:Cells, Cultured; D005260:Female; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000166:cytology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D013194:Staining and Labeling / Q000379:methods*; D013388:Succinimides / Q000737:chemistry*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides