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High-Content Quantification of Single-Cell Immune Dynamics.

Junkin M, Kaestli AJ, Cheng Z, Jordi C, Albayrak C, Hoffmann A, Tay S - Cell Rep (2016)

Bottom Line: Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems.We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically.Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.

No MeSH data available.


Related in: MedlinePlus

Multiplexed Endpoint Measurements of Single Cells(A) Multiplexed detection of cytokine release. Measuring 2 hr of secretion of the indicated cytokines in response to continuous exposure to 500 ng ml−1 LPS is shown. The Inset shows the multiple bead types loaded into the binding chamber, The scale bar represents 50 μm.(B) On-chip staining. Single 3T3 cells were isolated inside chambers, cultured, then fixed and stained on the chip for actin (red). Cells additionally possessed a nuclear marker (H2B-GFP). The scale bar represents 50 μm.(C) Single-cell harvesting from chip and clonal expansion. Images show initial single-cell harvesting into a 96-well plate on (day 1) and subsequent expansion (day 10) of a 3T3 fibroblast previously isolated and cultured inside the microfluidic device.(D) Single-cell gene expression for cells isolated, cultured on, and harvested from the microfluidic device. The data are the absolute copy number of Gapdh for KL-25 hybridoma cells (left) and CD147 for Jurkat cells (right), measured with digital PCR. Bulk measurements are shown as round dots. Error bars are the SD of bulk measurements. Lower figures show binned data and fits to gamma distributions.See also Supplemental Information.
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fig3: Multiplexed Endpoint Measurements of Single Cells(A) Multiplexed detection of cytokine release. Measuring 2 hr of secretion of the indicated cytokines in response to continuous exposure to 500 ng ml−1 LPS is shown. The Inset shows the multiple bead types loaded into the binding chamber, The scale bar represents 50 μm.(B) On-chip staining. Single 3T3 cells were isolated inside chambers, cultured, then fixed and stained on the chip for actin (red). Cells additionally possessed a nuclear marker (H2B-GFP). The scale bar represents 50 μm.(C) Single-cell harvesting from chip and clonal expansion. Images show initial single-cell harvesting into a 96-well plate on (day 1) and subsequent expansion (day 10) of a 3T3 fibroblast previously isolated and cultured inside the microfluidic device.(D) Single-cell gene expression for cells isolated, cultured on, and harvested from the microfluidic device. The data are the absolute copy number of Gapdh for KL-25 hybridoma cells (left) and CD147 for Jurkat cells (right), measured with digital PCR. Bulk measurements are shown as round dots. Error bars are the SD of bulk measurements. Lower figures show binned data and fits to gamma distributions.See also Supplemental Information.

Mentions: Measurement of single-cell secreted molecules is achieved by conducting on-chip bead-based fluorescent sandwich immunoassays upon the medium surrounding a cell. Prior to experiments, antibody functionalized beads are loaded into a series of storage chambers located in the lower half of the chip (Figure 1). Beads are retained during loading by 4-μm PDMS slits (Movie S3). When needed, they are moved via an on-chip peristaltic pump into a binding chamber (Figure 1C; Figure S1; Movie S3). During a measurement, the medium surrounding a cell is pumped into the binding chamber (Movie S4). The medium and beads are then sealed in the chamber to prevent loss of secreted molecules and are mixed by circular pumping that moves the secreted molecules over bead surfaces for efficient capture (Movies S5 and S6). After sufficient mixing for binding (Figure 2A), the chamber and beads are then rinsed and the beads are moved back to their holding areas. On-chip rinsing was critical in obtaining reproducible cytokine concentration measurements as it removes possible interfering molecules and excess antibodies between assay steps. Once all time point measurements have been conducted, detection antibodies for the sandwich assay are provided (Figure S1). When the entire assay is complete, beads are imaged and their fluorescent intensity is correlated to a calibration curve to calculate the number of cytokines released by a cell (Figure 2B). Calibrations are conducted on the chip, under identical conditions of temperature, humidity, and surface functionalization to account for factors such as affinity and cytokine diffusion present during measurements. Additionally, when multiple cytokine-specific beads are used together in a given chamber, multiplexed detection of cytokines from a single cell is possible (Figure 3A). This automated system allowed a 2-hr resolution for quantifying the concentration of single-cell secreted cytokines after stimulation with time-varying inflammatory molecules. The chip can additionally be reloaded with new beads after initial measurements to extend the measurement period if necessary.


High-Content Quantification of Single-Cell Immune Dynamics.

Junkin M, Kaestli AJ, Cheng Z, Jordi C, Albayrak C, Hoffmann A, Tay S - Cell Rep (2016)

Multiplexed Endpoint Measurements of Single Cells(A) Multiplexed detection of cytokine release. Measuring 2 hr of secretion of the indicated cytokines in response to continuous exposure to 500 ng ml−1 LPS is shown. The Inset shows the multiple bead types loaded into the binding chamber, The scale bar represents 50 μm.(B) On-chip staining. Single 3T3 cells were isolated inside chambers, cultured, then fixed and stained on the chip for actin (red). Cells additionally possessed a nuclear marker (H2B-GFP). The scale bar represents 50 μm.(C) Single-cell harvesting from chip and clonal expansion. Images show initial single-cell harvesting into a 96-well plate on (day 1) and subsequent expansion (day 10) of a 3T3 fibroblast previously isolated and cultured inside the microfluidic device.(D) Single-cell gene expression for cells isolated, cultured on, and harvested from the microfluidic device. The data are the absolute copy number of Gapdh for KL-25 hybridoma cells (left) and CD147 for Jurkat cells (right), measured with digital PCR. Bulk measurements are shown as round dots. Error bars are the SD of bulk measurements. Lower figures show binned data and fits to gamma distributions.See also Supplemental Information.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835544&req=5

fig3: Multiplexed Endpoint Measurements of Single Cells(A) Multiplexed detection of cytokine release. Measuring 2 hr of secretion of the indicated cytokines in response to continuous exposure to 500 ng ml−1 LPS is shown. The Inset shows the multiple bead types loaded into the binding chamber, The scale bar represents 50 μm.(B) On-chip staining. Single 3T3 cells were isolated inside chambers, cultured, then fixed and stained on the chip for actin (red). Cells additionally possessed a nuclear marker (H2B-GFP). The scale bar represents 50 μm.(C) Single-cell harvesting from chip and clonal expansion. Images show initial single-cell harvesting into a 96-well plate on (day 1) and subsequent expansion (day 10) of a 3T3 fibroblast previously isolated and cultured inside the microfluidic device.(D) Single-cell gene expression for cells isolated, cultured on, and harvested from the microfluidic device. The data are the absolute copy number of Gapdh for KL-25 hybridoma cells (left) and CD147 for Jurkat cells (right), measured with digital PCR. Bulk measurements are shown as round dots. Error bars are the SD of bulk measurements. Lower figures show binned data and fits to gamma distributions.See also Supplemental Information.
Mentions: Measurement of single-cell secreted molecules is achieved by conducting on-chip bead-based fluorescent sandwich immunoassays upon the medium surrounding a cell. Prior to experiments, antibody functionalized beads are loaded into a series of storage chambers located in the lower half of the chip (Figure 1). Beads are retained during loading by 4-μm PDMS slits (Movie S3). When needed, they are moved via an on-chip peristaltic pump into a binding chamber (Figure 1C; Figure S1; Movie S3). During a measurement, the medium surrounding a cell is pumped into the binding chamber (Movie S4). The medium and beads are then sealed in the chamber to prevent loss of secreted molecules and are mixed by circular pumping that moves the secreted molecules over bead surfaces for efficient capture (Movies S5 and S6). After sufficient mixing for binding (Figure 2A), the chamber and beads are then rinsed and the beads are moved back to their holding areas. On-chip rinsing was critical in obtaining reproducible cytokine concentration measurements as it removes possible interfering molecules and excess antibodies between assay steps. Once all time point measurements have been conducted, detection antibodies for the sandwich assay are provided (Figure S1). When the entire assay is complete, beads are imaged and their fluorescent intensity is correlated to a calibration curve to calculate the number of cytokines released by a cell (Figure 2B). Calibrations are conducted on the chip, under identical conditions of temperature, humidity, and surface functionalization to account for factors such as affinity and cytokine diffusion present during measurements. Additionally, when multiple cytokine-specific beads are used together in a given chamber, multiplexed detection of cytokines from a single cell is possible (Figure 3A). This automated system allowed a 2-hr resolution for quantifying the concentration of single-cell secreted cytokines after stimulation with time-varying inflammatory molecules. The chip can additionally be reloaded with new beads after initial measurements to extend the measurement period if necessary.

Bottom Line: Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems.We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically.Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.

No MeSH data available.


Related in: MedlinePlus