![]() Alternative approaches include strategies to genetically tag cells of interest with a fluorescent marker that allows for simultaneous identification of the cell type while performing the real time imaging analysis 17. Finally, when the proportion of a given cell type in a cell mixture is low, both the yield and purity of separated cells can be low 16, increasing the likelihood of contamination by unwanted cells. Furthermore, the binding of fluorescent antibodies to cell-specific surface markers that enables the separation process can inadvertently stimulate target cells, thereby affecting subsequent analysis. Detection of the expression of genetic markers of adult pancreatic cell types typically involves cell fixation 15. Specifically, fluid shear stress on cells during FACS separation can be both variable and much greater than occurs in vivo, causing variable impairment of both cell viability 11, 12 and cell functions such as gene expression 13 and cell cycle 14. One approach to addressing these challenges is to sort and purify cells prior to study using Fluorescence Activated Cell Sorting (FACS) 10, but this separation method can adversely affect cell function and viability. Achieving this goal, however, requires either that the cells are purified prior to study or that steps are taken beforehand to enable specific cell types to be identified within a complex cell mixture. Such challenges can be addressed through an approach to single cell functional assessment that permits statistical analysis of the distributions of the responses. ![]() Even homogeneous cell mixtures can be characterized by wide variability in individual cellular responses, the nature of which may be physiologically or pathophysiologically important to characterize 9. These examples highlight instances in which measures of bulk cell response are uninformative with respect to cell-specific behavior. The ability to discriminate between these selective drug effects requires high-throughput cellular analysis methods that are not currently available. Assessing cellular differences in drug toxicity within a given tissue preparation can also be confounded if, for example, a sparsely represented cell type, but not the major parenchymal cell type, is targeted and eliminated by the drug. Other examples include the need to identify and characterize cells isolated from primary tissues such as liver 2, 3, pancreatic islets 4, 5, brain 6, cardiomyocytes 7 or blood leukocytes 8. Differences in cell fate specification, inefficient transitions of a given cell phenotype through specific stages of development, and intrinsic heterogeneity existing within populations of progenitor cells 1 can each result in complex admixtures of many distinct cell types, and identifying and characterizing individual cell types in that mixture can be challenging. One example involves studies of directed differentiation of stem cells toward a given cell type of interest. ![]() This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.Ī common challenge in cell biology is the need to assess the functional attributes of isolated primary cells in heterogeneous cell mixtures. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. ![]() To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H 2O 2 and cAMP). Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Functional characterization of individual cells within heterogeneous tissue preparations is challenging. ![]()
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