Flow cytometry-based characterization of label-retaining stem cells following transplacental BrdU labelling.
A method to characterize and culture stem cells from neonate mouse epidermis after transplacental BrdU (bromo-deoxyuridine) administration is described. We have characterized stem cells by their properties viz. to retain BrdU label, adhere rapidly onto collagen-fibronectin substratum and express a specific biomarker beta-1-integrin. BrdU-labelled cells (detected using monoclonal antibody) constituted a sum of 18% of the total number of cells. The ability of freshly isolated keratinocytes [LRCs (label-retaining cells)] to bind to primary BrdU antibody or to pick up PI (propidium iodide) stain was distinguishable. Viable LRCs did not retain PI. Such cells, termed EpSC (epidermis stem cell), were PI negative and BrdU positive. EpSC constituted 6% of the total cell yield. Culture in low Ca2+ medium and susceptibility to differentiation in the presence of high Ca2+ levels further characterized the stem cells. This protocol is useful for studying transplacental carcinogenesis.
Environmental Carcinogenesis Laboratory, Indian Institute of Toxicology Research CSIR, Mahatma Gandhi Marg, PO Box 80, Lucknow 226001, India.
Shiv
S
Poojan
Sushil
S
Kumar
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015415:Biomarkers / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000008:administration & dosage* / Q000032:analysis / Q000276:immunology; D002118:Calcium / Q000494:pharmacology; D018929:Cell Culture Techniques; D002469:Cell Separation; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D000078404:Epidermal Cells; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000378:metabolism; D008431:Maternal-Fetal Exchange; D051379:Mice; D011247:Pregnancy; D011419:Propidium / Q000737:chemistry
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D005456:Fluorescent Dyes; D019012:Integrin beta1; D011419:Propidium; D001973:Bromodeoxyuridine; D002118:Calcium