Flow cytometry-based characterization of label-retaining stem cells following transplacental BrdU labelling

Abstract

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.

Disruption of Skin Stem Cell Homeostasis following Transplacental Arsenicosis; Alleviation by Combined Intake of Selenium and Curcumin

“…The multipotent adult stem cells were isolated from epidermis of neonate BALB/c mouse skin, and were cultured as described earlier [ 23 , 24 ]. In brief, the excised epidermis was digested enzymatically; keratinocytes were isolated and cultured in growth-promoting medium in a CO 2 incubator set at 5% CO 2 and 37°C.…”

“…After iAs exposures, the zero day old neonates were processed as earlier for stem cell isolation and FACS based characterization [ 21 , 23 – 24 ]. All experiments were approved by the CSIR-IITR IAEC (Institutional Animal Ethics Committee) according to CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Guidelines of Government of India.…”

“…wt., Sigma catalogue #B5002) was administered to female mice twice a day for four days before mating. The LRKs were examined in neonatal epidermis keratinocyte isolates after a chase period of 28 days [ 24 ]. After excising skin and stripping off epidermis, EpASCs were isolated and cultured ex vivo as described before.…”

Recovery and Cultivation of Keratinocytes From Shipped Mouse Skin

“…HSP27, HSP60, and HSP70 are elevated when the mouse keratinocytes exposed to heat or cold shock (Huang et al, ). Beta‐1 integrin/CD29 is expressed in the isolated mouse keratinocytes actively incorporating BrdU on the growing phase (Poojan and Kumar, ). In the control, non‐shipped cells (Figs.…”

“…Murine keratinocyte culture from neonatal skin (Yuspa et al, 1970;Lichti et al, 2008;Poojan et al, 2011) provides an approach to investigate the role of specific genes by using keratinocytes from transgenic mice. Often, the skin from the transgenic mice is collected and the keratinocytes are isolated in the investigator's lab.…”