Limits...
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

High-Content Profiling of Single-Cell Responses to Dynamic Immune Inputs(A) Top: fluorescence and brightfield image series of a single cell over the course of LPS stimulation experiment. The scale bar represents 20 μm. Bottom: corresponding data from this cell for NF-κB (RelA) activity, TNF secretion, morphology, and migration responses are shown. LPS stimulation depicted by the tan bar was 500 ng ml−1 for 2 hr.(B) RelA translocation and TNF secretion for distinct LPS input dynamics. The single 2-hr pulse of LPS (left), continuous LPS exposure (center), and 8-min pulses of LPS every 2 hr (right) are shown. The bars above heatmaps depict stimulation profiles, and scale bars depict normalized nuclear RelA and molecules of TNF. Continuous stimulation leads to significantly increased cell-to-cell variability and oscillatory dynamics in TNF secretion compared to transient or pulsed stimulation. RelA is normalized to initial nuclear fluorescence intensity of each cell prior to stimulation.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835544&req=5

fig4: High-Content Profiling of Single-Cell Responses to Dynamic Immune Inputs(A) Top: fluorescence and brightfield image series of a single cell over the course of LPS stimulation experiment. The scale bar represents 20 μm. Bottom: corresponding data from this cell for NF-κB (RelA) activity, TNF secretion, morphology, and migration responses are shown. LPS stimulation depicted by the tan bar was 500 ng ml−1 for 2 hr.(B) RelA translocation and TNF secretion for distinct LPS input dynamics. The single 2-hr pulse of LPS (left), continuous LPS exposure (center), and 8-min pulses of LPS every 2 hr (right) are shown. The bars above heatmaps depict stimulation profiles, and scale bars depict normalized nuclear RelA and molecules of TNF. Continuous stimulation leads to significantly increased cell-to-cell variability and oscillatory dynamics in TNF secretion compared to transient or pulsed stimulation. RelA is normalized to initial nuclear fluorescence intensity of each cell prior to stimulation.

Mentions: Quantification of the macrophage input-output system was undertaken by supplying cells with a series of dynamic immune stimulatory scenarios. Beads custom functionalized to capture TNF molecules were loaded into the chip, and single mouse macrophages (RAW 264.7) were then seeded into the 40 cell chambers as described. Prior to stimulation, baseline measurements of TNF secretion were conducted by supplying medium without any stimulant to provide insight to the cell state before stimulus. Cells were then stimulated with various dynamic inputs, and every 2 hr the surrounding medium was transferred to binding chambers and mixed with beads for the first part of the sandwich assay. Cells were not serum starved before or during stimulations. The dynamical single-cell data gathered are shown in Figure 4A, including recording of single-cell TNF secretion, transcription factor activity (nuclear localization of NF-κB), morphology, and cell migration.


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)

High-Content Profiling of Single-Cell Responses to Dynamic Immune Inputs(A) Top: fluorescence and brightfield image series of a single cell over the course of LPS stimulation experiment. The scale bar represents 20 μm. Bottom: corresponding data from this cell for NF-κB (RelA) activity, TNF secretion, morphology, and migration responses are shown. LPS stimulation depicted by the tan bar was 500 ng ml−1 for 2 hr.(B) RelA translocation and TNF secretion for distinct LPS input dynamics. The single 2-hr pulse of LPS (left), continuous LPS exposure (center), and 8-min pulses of LPS every 2 hr (right) are shown. The bars above heatmaps depict stimulation profiles, and scale bars depict normalized nuclear RelA and molecules of TNF. Continuous stimulation leads to significantly increased cell-to-cell variability and oscillatory dynamics in TNF secretion compared to transient or pulsed stimulation. RelA is normalized to initial nuclear fluorescence intensity of each cell prior to stimulation.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig4: High-Content Profiling of Single-Cell Responses to Dynamic Immune Inputs(A) Top: fluorescence and brightfield image series of a single cell over the course of LPS stimulation experiment. The scale bar represents 20 μm. Bottom: corresponding data from this cell for NF-κB (RelA) activity, TNF secretion, morphology, and migration responses are shown. LPS stimulation depicted by the tan bar was 500 ng ml−1 for 2 hr.(B) RelA translocation and TNF secretion for distinct LPS input dynamics. The single 2-hr pulse of LPS (left), continuous LPS exposure (center), and 8-min pulses of LPS every 2 hr (right) are shown. The bars above heatmaps depict stimulation profiles, and scale bars depict normalized nuclear RelA and molecules of TNF. Continuous stimulation leads to significantly increased cell-to-cell variability and oscillatory dynamics in TNF secretion compared to transient or pulsed stimulation. RelA is normalized to initial nuclear fluorescence intensity of each cell prior to stimulation.
Mentions: Quantification of the macrophage input-output system was undertaken by supplying cells with a series of dynamic immune stimulatory scenarios. Beads custom functionalized to capture TNF molecules were loaded into the chip, and single mouse macrophages (RAW 264.7) were then seeded into the 40 cell chambers as described. Prior to stimulation, baseline measurements of TNF secretion were conducted by supplying medium without any stimulant to provide insight to the cell state before stimulus. Cells were then stimulated with various dynamic inputs, and every 2 hr the surrounding medium was transferred to binding chambers and mixed with beads for the first part of the sandwich assay. Cells were not serum starved before or during stimulations. The dynamical single-cell data gathered are shown in Figure 4A, including recording of single-cell TNF secretion, transcription factor activity (nuclear localization of NF-κB), morphology, and cell migration.

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