hoechst side population assay

Copyright © 2006 by The American Society of Hematology 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.

Hematopoietic stem cells do not engraft with absolute efficiencies.

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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. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. 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. Flow cytometry of the side population: tips and tricks.

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). Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the “side population” (SP). Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Camargo FD, Chambers SM, Drew E, McNagny KM, Goodell MA. Search for other works by this author on:

Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Zhou S, Schuetz JD, Bunting KD, et al. 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, … 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 ]. The side population (SP) assay has been utilized as a method for isolation and characterization of normal and cancer stem cells from a variety of tissues. 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. Fried J, Doblin J, Takamoto S, Perez A, Hansen H, Clarkson B. 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.

The Hoechst purification was established for murine hematopoietic stem cells (HSC) on normal C57Bl/6 bone marrow (NBM). 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. Search for other works by this author on: Blau HM, Brazelton TR, Weimann JM.

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 evolving concept of a stem cell: entity or function? Matsuzaki Y, Kinjo K, Mulligan RC, Okano H. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells.

Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA.

I ntroduction . Scharenberg CW, Harkey MA, Torok-Storb B. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. 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. By continuing you agree to the Copyright © 2020 Elsevier B.V. or its licensors or contributors.

Sales-Pardo I, Avendaño A, Martinez-Muñoz V, et al. 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. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Uchida N, Fujisaki T, Eaves AC, et al. 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. Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. Isolation and functional properties of murine hematopoietic stem cells that are repopulating in vivo. This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so‐called side population. We suggest that initial experiments be performed using this marrow exactly as we describe in order to establish the procedure in your laboratory and to definitively identify the side population (SP) on the flow cytometer. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. the Hoechst 33342 dye is specific for DNA binding, ribonuclease treatment is not needed to avoid nonspecific RNA staining. Search for other works by this author on: