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Measurement of S‐phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis
Author(s) -
Verdoodt Freija,
Willems Maxime,
Dhondt Ineke,
Houthoofd Wouter,
Bert Wim,
Vos Winnok H.
Publication year - 2012
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1042/cbi20120187
Subject(s) - colocalization , biology , planarian , flatworm , stem cell , microbiology and biotechnology , regeneration (biology) , autofluorescence , population , live cell imaging , cell , anatomy , zoology , fluorescence , genetics , optics , medicine , physics , environmental health
Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo . These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated T s (S‐phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano . A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity‐based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object‐based colocalization approach was devised to score the relative number of double‐positive cells. Using this approach, T s (S‐phase duration) in the main population of neoblasts was ∼13 h. During early regeneration, no significant change in T s was observed.