Flow cytometry components

DCIA has been used to label numerous proteins and other biomolecules, including phospholipids (Silvius et al., 1987), to study the interaction of mRNA with the 30S ribosomal subunit (Czworkowski et al., 1991), in the investigation of cellular thiol components by flow cytometry (Durand and Olive, 1983), in the detection of carboxylate compounds using peroxyoxalate chemiluminescence (Grayeski and DeVasto, 1987), and for general sulfhydryl labeling (Sippel, 1981). 

Covalent attachment of antibody molecules to liposomes can provide a targeting capacity to the vesicle that can modulate its binding to specific antigenic determinants on cells or to molecules in solution. Antibody-bearing liposomes may possess encapsulated components that can be used for detection or therapy (Figure 22.17). For instance, fluorescent molecules encapsulated within antibody-targeted vesicles can be used as imaging tools or in flow cytometry... 

Fluorochromes are stains that interact directly with cellular components, or are used to form conjugates with antibodies or ligands, yielding fluorescent reporter molecules. O Table 13-1 lists a selection of fluoro- D Table 13-1 Fluorochromes for flow cytometry ... 

Flow cytometry (FCM) is a high-precision technique for rapid analysis and sorting of cells and particles. In theory, it can be used to measure any cell component, provided that a fluorescent tracer is available that reacts specifically and stoichiometrically with that constituent. The technique provides statistical accuracy, reproducibility, and sensitivity. 

These MHG class I alterations have been evaluated by the analysis of the panel of tumor samples with immunohistochemistry or flow cytometry in disrupted tumor cell suspensions as well as established tumor cell lines using a series of mAbs directed against HLA class I monomorphic, HLA-A or -B locus-specific, HLA-allelic epitopes or against various APM components including TAPI, TAP2, tpn and the different LMP subunits. The results demonstrated that rates of HLA class I and APM component losses in tumor cell lines strongly varied between the different tumor types analyzed. 

Among the items that have been measured are vitality, intracellular pH, DNA and RNA content, and specific plasmids [77,408]. Besides nucleic acids [204], other intracellular components can also be analyzed, e.g. storage materials [2, 82,294], enzymes and protein content [6,338], or the cell size [60,61]. The physiological state can also be rapidly assessed [331]. Furthermore, this technique allows the separation of certain cells using a cell sorter, e.g. for strain improvement [28]. The flow cytometry technique has also been used in connection with molecular probes for identification and viability determination of microbial communities [98]. This application of viability estimation is becoming increasingly important [63, 136, 188, 454]. Unfortunately, the equipment is expensive and most of the measurements are tricky and laborious and not well designed for on-line application. 

Jayat and Ratinaud [190] discuss the advantages of multi-parametric analyses taking DNA and other cellular components simultaneously into account see also, e.g. [73, 261]. Flow cytometry is also useful for identification of (new) species [78] or for biomass estimation [299,349]. 

Hewitt et al. [167] have exploited flow cytometry to quantify the impact of fluid mechanical stress on bacterial cultures. A modified technique, called the slit-scan method, allows the determination of cell shapes and of intracellular location of stained components [34]. Image cytometry and fluorescence microscopy are variants for determination of the volume growth of cells or morphology changes and seem to have become increasingly important [60,382,461 ]. 

In flow cytometry, measurements are made as cells or particles suspended in a fluid pass through the apparatus in single file. This allows the characterization and measurement (multiparametric) of fimctional aspects of single cells or particles, and electrical or mechanical sorting of cells/particles into distinct populations. In order to understand the basis of flow cytometry it is important to consider the key components comprising a flow cytometer (see Figure 6.9). 

Testing can be performed on genomic DNA isolated from the entire ceU population of the specimen. Alternatively, the sample can be sorted by flow cytometry or immunorosetting to assess engraftment of specific ceil lineages. For example, engraftment of the T-cell versus non-T-cell components can be assessed after flow cytometry sorting based on expression of the CD3 surface molecule, a T-ceU marker. 

Ogino T, WangX, Ferrone S (2003) Modified flow cytometry and cell-ELISA methodology to detect HLA class I antigen processing machinery components in cytoplasm and endoplasmic reticulum. J Immunol Methods 278 33-44... 

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