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Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. We discuss the SP assay and its applications in stem cell biology, with an emphasis on the technical challenges related to sample preparation, data acquisition, analysis, and interpretation. ABCG2‐linked transporter function has been used extensively in the detection and FACS isolation of pluripotential “side populations” [SPs; ] by virtue of the rapid energy‐dependent efflux of the DNA minor groove‐binding UV‐excitable dye Hoechst 33342 and the modified fluorescence emission spectrum in such cells (5, 10, 12-17). In this study, we used flow cytometry and Hoechst 33342 dye efflux assay to isolate and characterize SP cells from six human lung cancer cell lines (H460, H23, HTB-58, A549, H441, and H2170). 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). Search for other works by this author on:
ScienceDirect ® is a registered trademark of Elsevier B.V.Critical Appraisal of the Side Population Assay in Stem Cell and Cancer Stem Cell ResearchCopyright © 2011 Elsevier Inc. All rights reserved.ScienceDirect ® is a registered trademark of Elsevier B.V. Zhou S, Schuetz JD, Bunting KD, et al. The “Side Population” (SP) discrimination assay is a flow cytometry method used to detect stem cells based on the dye efflux properties of ABC transporters. Fried J, Doblin J, Takamoto S, Perez A, Hansen H, Clarkson B. Effects of Hoechst 33342 on survival and growth of two tumor cell lines and on hematopoietically normal bone marrow cells. Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the “side population” (SP). The Side Population (SP) discrimination assay is based on the differential potential of cells to efflux the Hoechst dye via the ATP-binding cassette (ABC) family of transporter proteins expressed within the cell membrane.
1 The authors used the Hoechst ABCG2-based efflux assay to isolate primitive stem cells from murine bone marrow (BM). Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. We highlight the value of multicolor phenotyping, the impact of DNA ploidy, and the importance of distinguishing graft versus host cells for an appropriate SP discrimination. This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so‐called side population.
Transplantable hematopoietic stem cells in human fetal liver have a CD34(+) side population (SP) phenotype. Flow cytometry of the side population: tips and tricks. the Hoechst 33342 dye is specific for DNA binding, ribonuclease treatment is not needed to avoid nonspecific RNA staining.
Blau HM, Brazelton TR, Weimann JM. Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Keywords: Cancer stem cells, Side population, Fluorescent probes, ABC transporters, JC-1, Hoechst 33342 Background Metabolic alteration in cancer cells has been considered to be linked to cancer progression and drug resistance, and thus characterized intensively for a long time as a potential therapeutic target [ 1 ]. We have read with great interest the recent paper published in Blood by Camargo et al. The Hoechst side population (SP) technique is an important methodology for identifying stem cells and early progenitors in rodent, nonhuman primate, and human hematopoietic and nonhematopoietic tissues [1, 2, 3 – 4].In this method, the cell‐permeable DNA‐binding dye Hoechst 33342 is passively loaded into cells. Hematopoietic stem cells do not engraft with absolute efficiencies.
We have read with great interest the recent paper published in H33342 retention experiments use living cells, allowing the investigators to study an active functional process based on the differential efflux of Reduced accumulation of several fluorescent dyes (ie, rhodamine 123) has also been observed in stem cells, and increased dye efflux has been associated with long-term repopulating ability.
The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Search for other works by this author on:
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They demonstrate that side population (SP) cells with a Hoechst 33342 low-fluorescent profile (SP low fraction) have a higher clonogenic potential than the rest of the SP, … Isolation and functional properties of murine hematopoietic stem cells that are repopulating in vivo. I ntroduction . Matsuzaki Y, Kinjo K, Mulligan RC, Okano H. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. In addition to its use in fluorescence microscopy and image analysis, Hoechst 33342 is commonly used for flow cytometric applications, such as cell cycle analysis and stem cell side population identification. Copyright © 2006 by The American Society of Hematology Search for other works by this author on:
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