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Flow Cytometry of the Side Population (SP).

Curr Protoc Cytom. 2007 Jan;Chapter 9:Unit9.23

Authors: Petriz J

The side population (SP) has become an important hallmark for the definition of the stem-cell compartment, especially for the detection of Stem Cells and for their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34(-) and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, the protocol is difficult for most investigators to perform: first, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because analysis of the acquired data requires extensive expertise in flow cytometry to accurately detect the SP events. This unit is packed with helpful pointers to aid the user in obtaining the best possible results.

PMID: 18770857 [PubMed - in process]

Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP).

Curr Protoc Cytom. 2005 Nov;Chapter 9:Unit9.18

Authors: Goodell MA

This unit describes the use of Hoechst 33342 to identify and purify murine Hematopoietic Stem Cells, the so-called side population. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Hoechst is a DNA-binding dye. It has at least two binding modes that result in different spectral properties, allowing resolution of multiple populations by viewing fluorescence at two wavelengths simultaneously. The ability to discriminate SP cells is based on the differential efflux of the dye by a multi-drug-like transporter. Particular attention is given to the critical aspects of Hoechst concentration, cell concentration, staining time, and staining temperature.

PMID: 18770827 [PubMed - in process]

Differential staining of DNA and RNA.

Curr Protoc Cytom. 2004 Nov;Chapter 7:Unit7.3

Authors: Darzynkiewicz Z, Juan G, Srour EF

Cell cycle analysis by means of differential staining of RNA and DNA permits determination of RNA content, which in turn allows one to discriminate G(0) versus G(1) cells and to detect cell Differentiation. This unit presents two protocols for differential staining, one using the metachromatic dye acridine orange (AO) and the other a combination of pyronin Y (PY) and Hoechst 33342. Each method has its advantages and limitations, and the Hoechst-PY method is not applicable to single-laser instruments. A third protocol describes staining of viable cells to identify/sort hematopoietic stem cells, with an alternative that includes simultaneous immunostaining.

PMID: 18770805 [PubMed - in process]

Enumeration of CD34(+) Hematopoietic Stem and Progenitor Cells.

Curr Protoc Cytom. 2003 Aug;Chapter 6:Unit6.4

Authors: Sutherland DR, Keeney M, Gratama JW

CD34 is the first documented cell surface antigen whose expression within the hematopoetic system is restricted to stem and progenitor cells of all lineages. Transplantation of CD34(+) cells has created a need for accurate and rapid assessment of CD34(+) cell concentrations in peripheral blood as well as for the optimal timing of apheresis sessions to insure the harvest of sufficient CD34(+) cells. However, accurate enumeration of these cells represents a rare-event analysis, a demanding and somewhat complicated procedure. This comprehensive unit presents the clearest and most definitive methods yet available for CD34(+) enumeration by flow cytometry, evaluates a number of commercially available procedures, and provides invaluable supporting information and detailed analysis which we believe is found in no other publication. All the possible problems are discussed and examples provided, allowing the clinical hematology lab to be certain that analysis of CD34(+) cells is performed in an appropriate manner.

PMID: 18770780 [PubMed - in process]

Immunophenotypic Analysis of PNH Cells.

Curr Protoc Cytom. 2002 May;Chapter 6:Unit6.11

Authors: Richards SJ, Hillmen P

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem-cell disorder in which a Gene mutation results in cellular inability to synthesize the glycosylphosphatidylinositol (GPI) anchors needed to attach various proteins to the cell membrane. Traditional clinical laboratory PNH screening and diagnosis are based upon demonstration of increased erythrocyte susceptibility to lysis by activated complement. These methods are neither very specific nor very sensitive, and have largely been superseded by the rapid, sensitive, and specific flow cytometric analysis of GPI-linked antigen expression. The protocols in this unit cover immunophenotypic analysis of red blood cells, peripheral blood granulocytes, and peripheral blood monocytes, as well as a four-color modification for simultaneous analysis of peripheral blood granulocytes and monocytes.

PMID: 18770768 [PubMed - in process]

Handling, storage, and preparation of human blood cells.

Curr Protoc Cytom. 2001 May;Chapter 5:Unit5.1

Authors: McCoy JP

Human peripheral blood samples are probably the most common specimens submitted for flow cytometric analysis. Handling and preparation will vary depending on the cell lineage to be examined and the type of assay. This unit presents protocols for separating white cells by lysing erythrocytes, isolating mononuclear cells by trilineage separation, and assorted methods for isolating or enriching specific cell populations, including monocytes, neutrophils, T lymphocytes, B lymphocytes, NK cells, stem cells, and platelets. Keywords: flow cytometry; human blood; leukocyte preparation; mononuclear blood cells; cell population enrichment; cell population isolation; cell population purification.

PMID: 18770711 [PubMed - in process]