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. 2013 Feb 19;85(4):2548-56.
doi: 10.1021/ac400082e. Epub 2013 Feb 1.

High-throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity

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Free PMC article

High-throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity

Yao Lu et al. Anal Chem. .
Free PMC article

Abstract

Secreted proteins dictate a range of cellular functions in human health and disease. Because of the high degree of cellular heterogeneity and, more importantly, polyfunctionality of individual cells, there is an unmet need to simultaneously measure an array of proteins from single cells and to rapidly assay a large number of single cells (more than 1000) in parallel. We describe a simple bioanalytical assay platform consisting of a large array of subnanoliter microchambers integrated with high-density antibody barcode microarrays for highly multiplexed protein detection from over a thousand single cells in parallel. This platform has been tested for both cell lines and complex biological samples such as primary cells from patients. We observed distinct heterogeneity among the single cell secretomic signatures that, for the first time, can be directly correlated to the cells' physical behavior such as migration. Compared to the state-of-the-art protein secretion assay such as ELISpot and emerging microtechnology-enabled assays, our approach offers both high throughput and high multiplicity. It also has a number of clinician-friendly features such as ease of operation, low sample consumption, and standardized data analysis, representing a potentially transformative tool for informative monitoring of cellular function and immunity in patients.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
High-throughput multiplexed single cell secretomic assay. (a) Schematic illustration showing integration of a high-density antibody barcode array chip and a subnanoliter microchamber array chip for high-throughput multiplexed protein secretion assay at the single cell level. (b) Scanned fluorescence image showing high uniformity of protein loading across the entire barcode microarray (1″ × 2″). Fluorescently labeled bovine serum albumin (FITC-BSA) was used in this test. (c) Photograph stitched from a large number of individual pictures collected by an automated, motorized phase contrast microscope. Together it covers the entire subnanoliter microchamber chip that was loaded with human immune cells (U973). Scale bar 2mm. The first enlarged image shows a column of microchamber array (scale bar 300μm). The second enlarged image shows individual cells loaded in microchambers (scale bar 50μm).
Figure 2
Figure 2
Protein panel. (a) List of all 22 proteins assayed in single cell microchips and their functions in human physiology. (b) Titration curves obtained using recombinant proteins. 18 antibody pairs were validated and 4 others were left out in the titration curves due to the lack of working recombinants. Fluorescence intensity represents the original photon counts averaged from 16 spots for each protein. Error bars indicate 3 × SD.
Figure 3
Figure 3
Single-cell secretomic analysis on U87 cell lines. (a) Representative region of the scanned image showing the raw data of single cell secretomic measurement. Three subpanels on the right are optical micrograph, fluorescence image and overlay for 16 microchambers. (b) Heat map that shows the profile of 14 proteins secreted from 1278 single cells (U87). Each row is a single cell and each column corresponds to a protein of interest. (c) Scatter plots showing fluorescence intensity measured for six selected proteins (FGF, VEGF, MIF, IL-6, IL-8, MCP-1) versus the number of cells in a microchamber. (* P < 0.05, ** P < 0.01, *** P < 0.001) (d) Population kinetics for U87 cell line. Control (MEM medium), secretion supernatant from population at different time points (0 hr, 1 hr, 2 hr, 3 hr, 6 hr, 9 hr, 12 hr, 24 hr).
Figure 4
Figure 4
Correlation between protein secretion profiles and cellular migration for A549 cells. (a) Representative optical images showing three single cells (n=384) before (0h) and after (24h) protein secretion assay. (b) Scatter plot showing the fluorescence intensity corresponding to IL-8 secretion versus migration distance of individual cells (P < 0.05). (c) Scatter plot showing a similar analysis on MCP-1 (P = 0.14). (d) Scatter plot showing a similar analysis on IL-6 (P = 0.75). Each dot represents a single cell.
Figure 5
Figure 5
Single-cell secretomic analysis of primary tumor cells from patients. (a) Procedure for processing tissue specimens, preparation of single cell suspension and application of primary cells to the subnanoliter microchamber array chip. (b) Representative region of the scanned image for Patient 1. (c) and (d) Heat maps showing single-cell secretomic signatures of primary tumor cells from two patients (Patients 1 and 2), respectively. The data are presented as a result of non-supervised hierarchical clustering analysis. (e) Histogram plots of individual proteins measured on the sample from Patient 1. (e) Scatter plot matrices showing protein-protein correlation in single cells. Each subpanel is the scatter plot showing the level of a protein versus the other in all single cells measured. The proteins are indicated at the diagonal line. The correlation coefficient is computed as R via a linear regression analysis. The entire matrix is color-coded by red (positive correlation) and blue (negative correlation). The color intensity is proportional to the R value.

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