Immunocytochemistry flow cytometry

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Another example of a population study that uses antibodies is flow cytometry. Isolated cells must be dissociated from tissues or cultures and labeled with fluorescent antibodies specific for a subpopulation of the cells. In flow cytometry, cells pass rapidly past a detector that measures the amount of fluorescence for each cell. The size of cells and the amount of fluorescence can be plotted and analyzed. Even though this method makes use of antibodies, it is a population study because it determines the number of isolated cells bound to an antibody. Flow cytometry identifies different populations of isolated cells, but it cannot show the location of these labeled cell in tissues, which can be done only with immunocytochemistry. 

First, in flow cytometry, the individual cells are in the light path for milliseconds. The rapid flow combined with short exposure to light eliminates problems due to photobleaching. Thus, the fluorophore fluorescein, one of the least costly fluorophores available, works well because of the short exposure to light even though it photobleaches very rapidly. In immunocytochemistry, photobleaching is a major problem. 

Before using a fluorophore-labeled antibody from flow cytometry for immunocytochemistry, check the spectra of fluorescent labels to rule out the possibility of bleed-through. Remember that although flow cytometry and immunocytochemistry use fluorescent dyes, the characteristics of these dyes are optimized for the technique and might not translate well when moved from flow cytometry to immunocytochemistry. 

In vitro, the capillary endothelial cells of the pancreas are still attached to the outer rim of whole islets postisolation and, with conditioned medium, may be coaxed to proliferate outward onto extracellular matrix-coated plastic plates for further mechanistic studies and functional analyses. The isolated capillary endothelial cells can be characterized by examining a-1 proteinase inhibitor and nephrin expression via immunocytochemistry or flow cytometry (Zanone et al., 2008). Not only do these endothelial cells play a role in drug candidate-induced effects on insulin secretion and glucose regulation of islets, but they also act as the regulators of leukocyte recruitment into the islets. The pancreatic microvasculature is therefore likely to play a role in the altered physiology of the endocrine and exocrine pancreas if affected by drug candidate exposure. 

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