Dataset Information

Integrated Platform for Monitoring Single-cell MAPK Kinetics in Computer-controlled Temporal Stimulations.

ABSTRACT: Extracellular response kinase (ERK) is one of the key regulator of cell fate, such as proliferation, differentiation and cell migration. Here, we propose a novel experimental pipeline to learn ERK kinetics by temporal growth factor (GF) stimulation. High signal-to-noise ratio of genetically encoded Fluorescence resonance energy transfer (FRET) biosensor enables to get a large number of single-cell ERK activity at each time point, while computer-controlled microfluidics fine-tune the temporal stimulation. Using this platform, we observed that static Epidermal growth factor (EGF) stimulation led to transient ERK activation with a significant cell-to-cell variation, while dynamic stimulation of 3' EGF pulse led to faster adaptation kinetics with no discrepancy. Multiple EGF pulses retriggered ERK activity with respect to frequency of stimulation. We also observed oscillation of ERK activity of each cell at basal state. Introducing of Mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, was not only dropping the average of basal activity for 7.5%, but also diminishing oscillatory behavior. Activity level raised up when inhibitor was removed, followed by transient peak of ERK kinetics. We expect this platform to probe Mitogen-associated protein kinase (MAPK) signaling network for systems biology research at single cellular level.

SUBMITTER: Ryu H

PROVIDER: S-EPMC6057930 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Integrated Platform for Monitoring Single-cell MAPK Kinetics in Computer-controlled Temporal Stimulations.

Ryu Hyunryul H Chung Minhwan M Song Jiyoung J Lee Sung Sik SS Pertz Olivier O Jeon Noo Li NL

Scientific reports 20180724 1

Extracellular response kinase (ERK) is one of the key regulator of cell fate, such as proliferation, differentiation and cell migration. Here, we propose a novel experimental pipeline to learn ERK kinetics by temporal growth factor (GF) stimulation. High signal-to-noise ratio of genetically encoded Fluorescence resonance energy transfer (FRET) biosensor enables to get a large number of single-cell ERK activity at each time point, while computer-controlled microfluidics fine-tune the temporal sti ...[more]

PMID: 30042437

Similar Datasets

Project description:Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes.

Project description:Plant phenotyping and production management are emerging fields to facilitate Genetics, Environment, & Management (GEM) research and provide production guidance. Precision indoor farming systems (PIFS), vertical farms with artificial light (aka plant factories) in particular, have long been suitable production scenes due to the advantages of efficient land utilization and year-round cultivation. In this study, a mobile robotics platform (MRP) within a commercial plant factory has been developed to dynamically understand plant growth and provide data support for growth model construction and production management by periodical monitoring of individual strawberry plants and fruit. Yield monitoring, where yield = the total number of ripe strawberry fruit detected, is a critical task to provide information on plant phenotyping. The MRP consists of an autonomous mobile robot (AMR) and a multilayer perception robot (MPR), i.e., MRP = the MPR installed on top of the AMR. The AMR is capable of traveling along the aisles between plant growing rows. The MPR consists of a data acquisition module that can be raised to the height of any plant growing tier of each row by a lifting module. Adding AprilTag observations (captured by a monocular camera) into the inertial navigation system to form an ATI navigation system has enhanced the MRP navigation within the repetitive and narrow physical structure of a plant factory to capture and correlate the growth and position information of each individual strawberry plant. The MRP performed robustly at various traveling speeds with a positioning accuracy of 13.0 mm. The temporal-spatial yield monitoring within a whole plant factory can be achieved to guide farmers to harvest strawberries on schedule through the MRP's periodical inspection. The yield monitoring performance was found to have an error rate of 6.26% when the plants were inspected at a constant MRP traveling speed of 0.2 m/s. The MRP's functions are expected to be transferable and expandable to other crop production monitoring and cultural tasks.

Project description:Spheroids are widely applied as building blocks for biofabrication of living tissues, where they exhibit spontaneous fusion toward an integrated structure upon contact. Tissue fusion is a fundamental biological process, but due to a lack of automated monitoring systems, the in-depth characterization of this process is still limited. Therefore, a quantitative high-throughput platform was developed to semi-automatically select doublet candidates and automatically monitor their fusion kinetics. Spheroids with varying degrees of chondrogenic maturation (days 1, 7, 14, and 21) were produced from two different cell pools, and their fusion kinetics were analyzed via the following steps: (1) by applying a novel spheroid seeding approach, the background noise was decreased due to the removal of cell debris while a sufficient number of doublets were still generated. (2) The doublet candidates were semi-automatically selected, thereby reducing the time and effort spent on manual selection. This was achieved by automatic detection of the microwells and building a random forest classifier, obtaining average accuracies, sensitivities, and precisions ranging from 95.0% to 97.4%, from 51.5% to 92.0%, and from 66.7% to 83.9%, respectively. (3) A software tool was developed to automatically extract morphological features such as the doublet area, roundness, contact length, and intersphere angle. For all data sets, the segmentation procedure obtained average sensitivities and precisions ranging from 96.8% to 98.1% and from 97.7% to 98.8%, respectively. Moreover, the average relative errors for the doublet area and contact length ranged from 1.23% to 2.26% and from 2.30% to 4.66%, respectively, while the average absolute errors for the doublet roundness and intersphere angle ranged from 0.0083 to 0.0135 and from 10.70 to 13.44°, respectively. (4) The data of both cell pools were analyzed, and an exponential model was used to extract kinetic parameters from the time-series data of the doublet roundness. For both cell pools, the technology was able to characterize the fusion rate and quality in an automated manner and allowed us to demonstrate that an increased chondrogenic maturity was linked with a decreased fusion rate. The platform is also applicable to other spheroid types, enabling an increased understanding of tissue fusion. Finally, our approach to study spheroid fusion over time will aid in the design of controlled fabrication of "assembloids" and bottom-up biofabrication of living tissues using spheroids.

Dataset Information

Integrated Platform for Monitoring Single-cell MAPK Kinetics in Computer-controlled Temporal Stimulations.

Publications

Integrated Platform for Monitoring Single-cell MAPK Kinetics in Computer-controlled Temporal Stimulations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets