Cell monitoring Flow cytometry

Corporeau, C. and Auffret, M., In situ hybridisation for flow cytometry a molecular method for monitoring stress-gene expression in hemolymph cells of oysters, Aquatic Toxicol., 64, 427, 2003. 

Analytical flow cytometry offers a rapid and facile means of monitoring cellular receptor content. For example, multiparameter flow cytometry techniques were used to monitor expression of GABAa receptor subunits during neurogenesis in embryonic rat brain (Marie et al., 2001). The content of the cell surface p75 neurotrophin receptor was measured in a heterogeneous population of mouse dorsal root sensory neurons, from which high and low p75 subsets were subsequently isolated by cell sorting (Barrett et al., 1998). 

Laser microbeams offer several advantages over other fluorescence excitation techniques. In spectrofluorometry, observations are often made on a population of cells in a cuvette, resulting in a combined signal that lacks information about individual cellular responses. In flow cytometry, many individual cells are measured, but there is no temporal resolution since each cell is observed only once, and there is no spatial resolution since the entire cell is illuminated as it passes through the laser beam (see Chapter 30). In conventional fluorescence microscopy, individual cells can be monitored over time, and information about the two-dimensional spatial distribution of fluorescence can be obtained. However, some samples may be more susceptible to photobleaching by the arc lamps used for excitation, and the temporal resolution is limited to video-rate data acquisition (30 frames/s) (see Chapter 14). 

Fluorescence excitation with a laser microbeam allows for a smaller region to be illuminated. Monitoring fluorescence with a sensitive photomultiplier tube also permits the use of lower intensities of irradiation for shorter periods of time. Therefore, unwanted photobleaching can be significantly reduced. If the spot size is adjusted to illuminate an entire cell, information analogous to spectrofluorometry or flow cytometry can be obtained on an individual cell basis with a high degree of temporal resolution. If the spot size is smaller than the cell, similar information can be obtained from a particular location within the cell. 

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... 

The difficulties of characterizing the cell physiological properties can explain the difficulties in applying these models and why they have not been widely used in the solution of biochemical engineering problems. On the other hand, the advances in monitoring, especially in flow cytometry, and in computer science development, that increase the capacity for model... 

While these examples illustrate the role of flow cytometry in bioprocess monitoring, the analyses have been conducted off-line thus making their use in bioprocess control impractical. Recently, a portable flow cytometer - the Microcyte - [148] has been described, which due to its small size and lower cost (compared to conventional machines) allows flow cytometry to be used as an at-line technique [149]. Ronning showed that this instrument had a role to play in the determination of viability of starter cultures and during fermentation. The physiological status of each individual cell is likely to be an important factor in the overall productivity of the culture and is therefore a key parameter in optimising production conditions. 

Fig. 7.4 (a) Dose-response of the lamellarin M-induced mitochondrial depolarization in P388 cells, (b) Monitoring of the mitochondrial membrane potential (A rm) by realtime flow cytometry, using functional mitochondria isolated from P388 cells and the fluorescent probe JC-1. 

However, the flow cytometers are bulky and expansive, and are available only in large reference laboratories. In addition, the required sample volumes are quite large, usually in the 100 pL range. Many clinical applications require frequent blood tests to monitor patients status and the therapy effectiveness. It is highly desirable to use only small amount of blood samples Ifom patients for each test. Furthermore, it is highly desirable to have affordable and portable flow cytometry instruments for field applications, point-of-care applications and applications in resource-limited locations. To overcome these drawbacks and to meet the increasing needs for versatile cellular analyses, efforts have been made recently to apply microfluidics and lab-on-a-chip technologies to flow cytometric analysis of cells. 

For cell-based Nab bioassays, it is critical to assess the stability of the cell line over time (passages) and through cryopreservation. Stability assessment should be performed by comparing assay controls such as concentration response curves or neutralizing capacity of the positive control (PC). Additional parameters, such as cell growth, appearance, and viability may also aid in stability assessment. Further, monitoring receptor expression by a method such as flow cytometry may also be valuable. 

Krutzik PO, Nolan GP. Intracellular phospho-protein staining techniques for flow cytometry monitoring single cell signahng events. Cytometry A 2003 55 61-70. 

The membrane potential of individual cells can be monitored with a fluorescence microscope. For this purpose, however, it is preferable to use a permeable redistribution dye with spectral characteristics that have minimal environmental sensitivity. Thus, the fluorescence intensity will reflect the degree of Nemstian accumulation of dye only and can, therefore, be readily interpreted. The plasma membrane potential can be distinguished from the organelle membrane by simply using the microscope to identify appropriate regions of the cell (44). Rhodamine-123 (Chart III) was introduced as a mitochondrial stain by Chen and co-workers (45-47) it has been used largely in qualitative studies of mitochondrial membrane potential and has been especially effective in flow cytometry applications. 

Xia, Z., E-L. Appelkvist, J. W. Depierre, and L. Nassberger. 1997. Tricyclic antidepressant-induced lipidosis in human monocytes in vitro as well as monocyte derived cell line, as monitored by spectrofluorimetry and flow cytometry after staining with Nile red. Biochemical Pharmacology 53 1521-1532. 

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