A method for high-throughput functional imaging of single cells within heterogeneous cell preparations

Abstract

Functional characterization of individual cells within heterogeneous tissue preparations is challenging. 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. 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 (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. 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. 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. 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.

Cytometry of Reaction Rate Constant: Measuring Reaction Rate Constant in Individual Cells To Facilitate Robust and Accurate Analysis of Cell-Population Heterogeneity

“…Robustness and accuracy of our kinetic analysis were studied in comparison to those of the common nonkinetic analysis of the same data set. A kinetic curve utilized for finding k MDR (see Figure ) was also used to calculate a nonkinetic measure of MDR transport, a relative decrease of fluorescence intensity, ( I 1 – I 2 )/ I 1 , − and construct the nonkinetic histogram “number of cells versus ( I 1 – I 2 )/ I 1 ”. The value of ( I 1 – I 2 )/ I 1 corresponds to a fraction of the MDR substrate extruded from the cell.…”

Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells

“…Traces of a few individual cells showing these heterogenous responses are shown in Figure S6 . However, only 20–30% of our cells showed consistent responses to rises in glucose, likely because of the heterogenous nature of INS1-832/13 cells [ 56 , 57 , 58 , 59 , 60 ], which can exhibit quite variable responses to glucose. We also noted that in some INS1-832/13 cells, the mitochondria exhibited robust Ca 2+ oscillations, as can be seen in Figure 3 c’s lower panel.…”

Twenty years of islet-on-a-chip: microfluidic tools for dissecting islet metabolism and function

“…Many islet-ona-chips have been used to image the Ca 2+ -activity of pancreatic islets using a wide range of dyes and genetically encoded sensors. 27,61,84,86,87 One common strategy is to simply incubate islets with Ca 2+ -sensitive dyes (e.g., Fluo-4, Fura 2, and Cal-520) made membrane permeable by the addition of acetoxymethyl (AM) ester. These AM-dyes are hydrolyzed by esterases once inside the cell, retaining them inside the cell and making them Ca 2+ -responsive.…”

“…Neal et al designed a custom-gridded glass coverslip for the commercially available FCS2 fluidics chamber. 84 Rat pancreatic islets were dispersed and the cells were seeded onto the coverslip for real-time NAD(P)H imaging and Ca 2+ -influx imaging (Fig. 7B, left).…”