Flow cytometry importance

Immunophenotyping by flow cytometry has taken on an increasingly important role in the diagnosis of leukemia. Owing to the ease of application, sensitivity, and quantifiable results, flow cytometry is the preferred method for lineage assignment.7 This approach takes advantage of the development of monoclonal antibodies... 

Darvey, H. M. Kell, D. B. Flow cytometry and cell sorting of heterogenous microbial populations the importance of single cell analysis. Microbiol. Rev. 1997, 60, 641-696. 

Follow-on studies are also recommended as needed. These include determination of potential test article effects on blood or tissue immunophenotypes (by flow cytometry or immunohistochemistry), natural killer cell, macrophage, or neutrophil function, host resistance to infection or tumors, and cell-mediated immunity. The important issue in all of these guidelines is this do not ignore signs of immunotoxicity, and assess these findings when observed. 

The above examples show that a very important criterion in the choice of a probe is its sensitivity to a particular property of the microenvironment in which it is located (e.g. polarity, acidity, etc.). On the other hand, insensitivity to the chemical nature of the environment is preferable in some cases (e.g. in fluorescence polarization or energy transfer experiments). Environment-insensitive probes are also better suited to fluorescence microscopy and flow cytometry. 

Increasing the simplicity and cost efficiency of algal tests has been an important research activity in recent years. [134,135] New tests procedures involve the apphcation of flow cytometry, microplate techniques, and immobilized algae [135-140]. 

Indeed, TCA (42) at a concentration of 10 Xg/mL, has been shown to elevate levels of ROS, as measured by flow cytometry. Consistent with earlier observations regarding structure-activity relationships, Me-TCA (44) showed 3-fold induction of ROS while dihydro-TCA (43) had no effect on the cellular levels of ROS.It is noteworthy that parthenolide (45), a sesquiterpene natural product structurally related to TCA, has previously been shown to increase the levels of ROS by glutathione depletion in hepatocellular carcinoma cell lines. In a separate study, parthenolide was able to inhibit DNA synthesis, cause cell cycle arrest, and induce apoptosis which are important mechanisms for controlling tumor growth. 

In this chapter, the analysis of leukocytes, one of the more important applications of flow cytometry, is discussed. It is an important application not because leukocytes are intrinsically more important than other types of cells but because they constitute a class of particles that, for several reasons, are ideally suited for analysis by flow cytometry and thus make very good use of the capabilities of the technique. Leukocytes therefore account for a large proportion of the material analyzed by flow cytometry in both hospital and research laboratories. They also serve as good examples for describing here some of the general procedures of cytometric protocol, particularly the art of gating and the requirements for controls. 

The only difficulty in using flow cytometry to monitor cell death is that, as mentioned in Chapter 3, dead cells have different scatter properties than living cells. In particular, because of their perforated outer membrane, they have a lower refractive index than living cells and therefore have forward scatter signals of lower intensity. For this reason, it is important not to use a gate or forward scatter threshold when analyzing a population for the proportion of dead and live cells. Any forward versus side scatter gate drawn around normal lymphocytes, for example, will always show most if not all of the cells... 

Apoptosis Apoptosis is an ordered, active process that brings about the death of a cell as an important part of the maintenance of organismal homeostasis. Apoptosis can be assayed, in flow cytometry, by, for example, looking at the expression of phosphatidylserine on the cell surface, by looking for nuclei with less-than-normal (sub-GO/Gl) amounts of DNA, and by looking for an increase in DNA fragment termini. 

Coaxial flow Coaxial flow is flow of a narrow core of liquid within the center of a wider stream. This type of flow is important in flow cytometry because it provides a means by which particles flowing through a relatively wide nozzle can be tightly confined in space, allowing accurate and stable illumination as they pass one by one through a light beam. 

Filter (2) A paper or nylon filter removes particles above a certain size from liquid. In flow cytometry, these filters are important for cutting down on background noise by removing small particles (<0.22 pm) from the sheath fluid and also for removing clumps from your sample (<35 pm). 

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

Laser Laser is an acronym for Light Amplification by. Stimulated Emission of Radiation. Lasers are important in flow cytometry because, as a result of their coherent output, they are a means of illuminating cells with a compact, intense light beam that will produce fluorescence signals that are as bright as possible over a short time period. 

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