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. 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. This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so‐called side population. Zhou S, Schuetz JD, Bunting KD, et al.
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.
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. These Bis-benzimides were originally developed by Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33,342nd compound made by the company. The Hoechst purification was established for murine hematopoietic stem cells (HSC) on normal C57Bl/6 bone marrow (NBM). Matsuzaki Y, Kinjo K, Mulligan RC, Okano H. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Camargo FD, Chambers SM, Drew E, McNagny KM, Goodell MA. the Hoechst 33342 dye is specific for DNA binding, ribonuclease treatment is not needed to avoid nonspecific RNA staining. Fried J, Doblin J, Takamoto S, Perez A, Hansen H, Clarkson B.
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.
Thus, the highest levels of multidrug transporters can be found in CD34For the SP assay, laser power and alignment, filter settings, sample collection, cell viability, We have described a method for isolating hematopoietic stem cells (HSCs) from murine bone marrow utilizing a Hoechst 33342 (Data on the toxicity of H33342 have been available for decades, as it is well known that H33342 is powerful inhibitor of DNA sythesis.Even though Hoechst staining does involve a great deal of viability loss in nonhematopoietic tissues,
Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. However, the SP phenotype may not be a common property of all stem cells. The evolving concept of a stem cell: entity or function?
Isolation and functional properties of murine hematopoietic stem cells that are repopulating in vivo. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. To improve the consistency and reliability of data between laboratories, we propose a set of recommendations for SP assay data reporting.We use cookies to help provide and enhance our service and tailor content and ads. Search for other works by this author on: Search for other works by this author on: I ntroduction . 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, … There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. Copyright © 2006 by The American Society of Hematology Hematopoietic stem cells do not engraft with absolute efficiencies. 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. Search for other works by this author on:
Search for other works by this author on: Irene Sales-Pardo, Ariadna Avendaño, Jordi Barquinero, Joan Carles Domingo, Pedro Marin, Jordi Petriz; The Hoechst low-fluorescent profile of the side population: clonogenicity versus dye retention. 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 ]. This is a functional assay that uses living cells, allowing investi- Hoechst is a DNA‐binding dye. 1 The authors used the Hoechst ABCG2-based efflux assay to isolate primitive stem cells from murine bone marrow (BM). 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).
Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the “side population” (SP). Goodell MA, Rosenzweig M, Kim H, et al.
Van Zant G, Fry CG. Uchida N, Fujisaki T, Eaves AC, et al.
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