This protocol describes the application of Hoechst 33342 (bisbenzimide, H 33342; Ho342) staining to identify and purify side-population (SP) stem cells from various human and murine tissue sources (Goodell et al., 1996, 1997; Storms et al., 2000; Jackson et al., 2001). This is a functional assay that uses living cells, allowing investigators to study an active biological process based on the di...

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane α-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably express...

OBJECTIVE The aim of this study was to verify the existence of a side population (SP) of cells in second-trimester amniotic fluid. MATERIALS AND METHODS Amniotic fluid samples (n = 35) were obtained, and the number and size of viable amniotic fluid cells (AFCs) were analyzed. Small AFCs (SAFCs) and large AFCs (LAFCs) were isolated using a sterile 10-microm pore size strainer. Hoechst 33342 dy...

The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segrega...

Cancer stem cells have been isolated from human gliomas and many other parenchymal tumors. It was previously assumed that many established malignant cell lines also contain a rare subpopulation of stem cells. This study was designed to investigate the fraction of cancer stem cells in the C6 glioma cell line using clonal and population analyses, rather than isolating methods, which are based on ...

The fluorescent stain Hoechst 33342, when injected i.v. into mice, has an LD50 of 300 gg-1. The stain exits rapidly from the blood, with a half-life of 110 sec following an injection of I0sg g-1, but remains bound within target cells, redistributing with a half-life longer than 2 h. This results in a gradient of drug binding outward from capillaries which can be used to estimate regional perfus...