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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.


On-Chip Bead Assay for Cytokine Secretion Measurement(A) Determination of needed mixing time. The capture of TNF on the bead surface with mixing inside a binding chamber is depicted. TNF was incubated with beads while undergoing mixing for different times before rinsing and completion of sandwich assay. Steady state is reached only after 2 hr of mixing.(B) On-chip calibration curve of TNF capture with bead-based sandwich assay. The detection limit was taken as three times the SD of negative control measurements (LOD ∼60,000 TNF molecules). Error bars are the standard error of the means.(C) Modeling the effect of mixing on assay performance. Random walk model simulated proportion of cytokines bound to bead surface after 1.5 hr inside a 2D channel. Curves show the effect of varying the diffusion coefficient, D, for TNF and for mixing.(D) Images from model depicting the benefit of mixing inside a 1000 × 100 μm chamber. Top images show a chamber where only diffusion is present. The area adjacent to the bead becomes depleted of cytokines (black dots) as they bind to the bead (red dot) over 1.5 hr, resulting in low capture efficiency. The lower images depict the channel when mixing is present. The rapid mixing achieved when medium is circularly pumped inside the binding chamber enables faster binding. Numbers in brackets indicate percentages of cytokine bound to bead.See also the Supplemental Experimental Procedures (“Random walk model for protein diffusion and capture”).
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fig2: On-Chip Bead Assay for Cytokine Secretion Measurement(A) Determination of needed mixing time. The capture of TNF on the bead surface with mixing inside a binding chamber is depicted. TNF was incubated with beads while undergoing mixing for different times before rinsing and completion of sandwich assay. Steady state is reached only after 2 hr of mixing.(B) On-chip calibration curve of TNF capture with bead-based sandwich assay. The detection limit was taken as three times the SD of negative control measurements (LOD ∼60,000 TNF molecules). Error bars are the standard error of the means.(C) Modeling the effect of mixing on assay performance. Random walk model simulated proportion of cytokines bound to bead surface after 1.5 hr inside a 2D channel. Curves show the effect of varying the diffusion coefficient, D, for TNF and for mixing.(D) Images from model depicting the benefit of mixing inside a 1000 × 100 μm chamber. Top images show a chamber where only diffusion is present. The area adjacent to the bead becomes depleted of cytokines (black dots) as they bind to the bead (red dot) over 1.5 hr, resulting in low capture efficiency. The lower images depict the channel when mixing is present. The rapid mixing achieved when medium is circularly pumped inside the binding chamber enables faster binding. Numbers in brackets indicate percentages of cytokine bound to bead.See also the Supplemental Experimental Procedures (“Random walk model for protein diffusion and capture”).

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)

On-Chip Bead Assay for Cytokine Secretion Measurement(A) Determination of needed mixing time. The capture of TNF on the bead surface with mixing inside a binding chamber is depicted. TNF was incubated with beads while undergoing mixing for different times before rinsing and completion of sandwich assay. Steady state is reached only after 2 hr of mixing.(B) On-chip calibration curve of TNF capture with bead-based sandwich assay. The detection limit was taken as three times the SD of negative control measurements (LOD ∼60,000 TNF molecules). Error bars are the standard error of the means.(C) Modeling the effect of mixing on assay performance. Random walk model simulated proportion of cytokines bound to bead surface after 1.5 hr inside a 2D channel. Curves show the effect of varying the diffusion coefficient, D, for TNF and for mixing.(D) Images from model depicting the benefit of mixing inside a 1000 × 100 μm chamber. Top images show a chamber where only diffusion is present. The area adjacent to the bead becomes depleted of cytokines (black dots) as they bind to the bead (red dot) over 1.5 hr, resulting in low capture efficiency. The lower images depict the channel when mixing is present. The rapid mixing achieved when medium is circularly pumped inside the binding chamber enables faster binding. Numbers in brackets indicate percentages of cytokine bound to bead.See also the Supplemental Experimental Procedures (“Random walk model for protein diffusion and capture”).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig2: On-Chip Bead Assay for Cytokine Secretion Measurement(A) Determination of needed mixing time. The capture of TNF on the bead surface with mixing inside a binding chamber is depicted. TNF was incubated with beads while undergoing mixing for different times before rinsing and completion of sandwich assay. Steady state is reached only after 2 hr of mixing.(B) On-chip calibration curve of TNF capture with bead-based sandwich assay. The detection limit was taken as three times the SD of negative control measurements (LOD ∼60,000 TNF molecules). Error bars are the standard error of the means.(C) Modeling the effect of mixing on assay performance. Random walk model simulated proportion of cytokines bound to bead surface after 1.5 hr inside a 2D channel. Curves show the effect of varying the diffusion coefficient, D, for TNF and for mixing.(D) Images from model depicting the benefit of mixing inside a 1000 × 100 μm chamber. Top images show a chamber where only diffusion is present. The area adjacent to the bead becomes depleted of cytokines (black dots) as they bind to the bead (red dot) over 1.5 hr, resulting in low capture efficiency. The lower images depict the channel when mixing is present. The rapid mixing achieved when medium is circularly pumped inside the binding chamber enables faster binding. Numbers in brackets indicate percentages of cytokine bound to bead.See also the Supplemental Experimental Procedures (“Random walk model for protein diffusion and capture”).
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.