Determining cell cycle stages by flow cytometry.
(Curr Protoc Cell Biol. 2001 May;Chapter 8:Unit 8.4.)
Darzynkiewicz Z, Juan G, Bedner E.
New York Medical College, Valhalla, New York, USA.
This unit describes assays used to determine the distribution of a population of
cells to the different stages of the cell cycle as analyzed by flow cytometry.
Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is
one of the most direct ways of staging the cells based on DNA content. These
staining procedures can be used with fixed cells or detergent-permeabilized
cells. Alternatively, live cells can be stained with the membrane-permeable
stain, Hoechst 33242, and sorted on the basis of DNA content for further culture
and characterization. When DNA content measurements are coupled with
immunostaining for cyclin expression, it is possible to reliably detect even more
stages in progression through the cell cycle.
Flow cytometry of apoptosis.
(Curr Protoc Cell Biol. 2004 Feb;Chapter 18:Unit 18.8.)
Pozarowski P, Grabarek J, Darzynkiewicz Z.
School of Medicine, Lublin, Poland.
Common methods applicable to flow cytometry make it possible to: (1) identify and
quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or
necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell
morphology and chromatin condensation, which occur during apoptosis, can be
detected by analysis with laser light beam scattering. Early events of apoptosis,
dissipation of the mitochondrial transmembrane potential and caspase activation,
can be detected using either fluorochrome reporter groups or appropriate
antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma
membrane can be detected by the binding of fluoresceinated annexin V. Another
apoptotic event, DNA fragmentation based on DNA content of cells with fractional
("sub-G1") or DNA strand-break labeling, TUNEL; or In Situ End Labeling, ISEL;.
Still another hallmark of apoptosis is the activation of tissue transglutaminase
(TGase), the enzyme that crosslinks protein and thereby makes them less
immunogenic. The major advantage of flow cytometry in these applications is that
it provides the possibility of multiparametric measurements of cell attributes.
Flow cytometry technique books (Biowww bookshelf)
flow cytometry FACS techniques
FACS kills culture cells from bone marrow (Forum)
Troubleshooting on FACS sorted cell culture