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Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses
Teste, Bruno, Champ, Jerome ; Londono-Vallejo, Arturo, Descroix, Stephanie, Malaquin, Laurent, Viovy, Jean-Louis, Draskovic, Irena, Mottet, Guillaume
Lab. Chip - 17, 3: 530-537 - DOI: 10.1039/c6lc01535b - 2017
Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.
FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.
Serra, M ; Pereiro, I; Yamada, A; Viovy, J. –L.; Descroix, S. Ferraro
Methods Mol Biol. - 1547 211-220 - doi: 10.1007/978-1-4939-6734-6_16 - 2017
The sealing of microfluidic devices remains a complex and time-consuming process requiring specific equipment and protocols: a universal method is thus highly desirable. We propose here the use of a commercially available sealing tape as a robust, versatile, reversible solution, compatible with cell and molecular biology protocols, and requiring only the application of manually achievable pressures. The performance of the seal was tested with regards to the most commonly used chip materials. For most materials, the bonding resisted 5 bars at room temperature and 1 bar at 95 °C. This method should find numerous uses, ranging from fast prototyping in the laboratory to implementation in low technology environments or industrial production.
A simple and low-cost chip bonding solution for high pressure, high temperature and biological applications Serra, M ; Pereiro, I; Yamada, A; Viovy, J. –L.; Descroix, S. Ferraro
Serra, M ; Pereiro, I; Yamada, A; Viovy, J. –L.; Descroix, S. Ferraro
Lab. Chip - 0.71111111111 629-634 - doi: 10.1039/c6lc01319h - 2017
The sealing of microfluidic devices remains a complex and time-consuming process requiring specific equipment and protocols: a universal method is thus highly desirable. We propose here the use of a commercially available sealing tape as a robust, versatile, reversible solution, compatible with cell and molecular biology protocols, and requiring only the application of manually achievable pressures. The performance of the seal was tested with regards to the most commonly used chip materials. For most materials, the bonding resisted 5 bars at room temperature and 1 bar at 95 °C. This method should find numerous uses, ranging from fast prototyping in the laboratory to implementation in low technology environments or industrial production.
Droplet Microfluidic and Magnetic Particles Platform for Cancer Typing.
Ferraro D, Champ J, Teste B, Serra M, Malaquin L, Descroix S, de Cremoux P, Viovy JL.
Lab. Chip - 1547 113-121 - doi: 10.1007/978-1-4939-6734-6_9. - 2017
Analyses of nucleic acids are routinely performed in hospital laboratories to detect gene alterations for cancer diagnosis and treatment decision. Among the different possible investigations, mRNA analysis provides information on abnormal levels of genes expression. Standard laboratory methods are still not adapted to the isolation and quantitation of low mRNA amounts and new techniques needs to be developed in particular for rare subsets analysis. By reducing the volume involved, time process, and the contamination risks, droplet microfluidics provide numerous advantages to perform analysis down to the single cell level.We report on a droplet microfluidic platform based on the manipulation of magnetic particles that allows the clinical analysis of tumor tissues. In particular, it allows the extraction of mRNA from the total-RNA sample, Reverse Transcription, and cDNA amplification, all in droplets.
Optimizing Hyperuniformity in Self-Assembled Bidisperse Emulsions
Joshua Ricouvier, Romain Pierrat, Rémi Carminati, Patrick Tabeling, and Pavel Yazhgur
Phys. Rev. Lett. - 119 208001 - doi.org/10.1103/PhysRevLett. - 2017
We study long range density fluctuations (hyperuniformity) in two-dimensional jammed packings of bidisperse droplets. Taking advantage of microfluidics, we systematically span a large range of size and concentration ratios of the two droplet populations. We identify various defects increasing long range density fluctuations mainly due to organization of local particle environment. By choosing an appropriate bidispersity, we fabricate materials with a high level of hyperuniformity. Interesting transparency properties of these optimized materials are established based on numerical simulations.
Microfluidic actuators based on temperature-responsive hydrogels
Joshua Ricouvier, Romain Pierrat, Rémi Carminati, Patrick Tabeling, Pavel Yazhgur
Phys. Rev. Lett. - 119 (20) 208001 - DOI:10.1103/PhysRevLett.119.208001 - 2017
We study long range density fluctuations (hyperuniformity) in two-dimensional jammed packings of bidisperse droplets. Taking advantage of microfluidics, we systematically span a large range of size and concentration ratios of the two droplet populations. We identify various defects increasing long range density fluctuations mainly due to organization of local particle environment. By choosing an appropriate bidispersity, we fabricate materials with a high level of hyperuniformity. Interesting transparency properties of these optimized materials are established based on numerical simulations.
Particle deposition kinetics of colloidal suspensions in microchannels at high ionic strength
Cesare M. Cejas, Fabrice Monti, Marine Truchet, Jean-Pierre Burnouf, and Patrick Tabeling
Langmuir - 33 (26) 6471–6480 - DOI: 10.1021/acs.langmuir.7b01394 - 2017
Despite its considerable practical importance, the deposition of real Brownian particles transported in a channel by a liquid, at small Reynolds numbers, has never been described at a comprehensive level. Here, by coupling microfluidic experiments, theory, and numerics, we succeed in unravelling the problem for the case of straight channels at high salinity. We discover a broad regime of deposition (the van der Waals regime) in which particle–wall van der Waals interactions govern the deposition mechanism. We determine the range of existence of the regime, for which we calculate the concentration profiles, retention profiles, and deposition kinetics analytically. The retention profiles decay as the inverse of the square root of the distance from the entry, and the deposition kinetics are given by the expression , where S is a dimensionless deposition function, A is the Hamaker constant, and ξL is a dimensionless parameter characterizing fluid flow properties. These findings are well supported by numerics. Experimentally, we find that the retention profiles behave as x–0.5±0.1 (where x is the distance from the channel entry) over three decades in scale, as predicted theoretically. By varying the flow conditions (speed, geometry, surface properties, and concentration) so as to cover four decades in ξL and taking the Hamaker constant as a free parameter, we accurately confirm the theoretical expression for the deposition kinetics. Operating in the van der Waals regime enables control of the deposition rates via surface chemistry. From a surface science perspective, working in the van der Waals regime enables us to measure the Hamaker constants of thousands of particles in a few minutes, a task that would take a much longer time to perform with standard AFM.
Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby Favoring the Induction of CD8 T Cell Responses.
Oyarce C, Cruz-Gomez S, Galvez-Cancino F, Vargas P, Moreau HD, Diaz-Valdivia N, Diaz J, Salazar-Onfray FA, Pacheco R6, Lennon-Dumenil AM, Quest AFG, Lladser A.
Front Immunol - 13;8 1794 - doi: 10.3389/fimmu.2017.01794 - 2017
Dendritic cell (DC) trafficking from peripheral tissues to lymph nodes (LNs) is a key step required to initiate T cell responses against pathogens as well as tumors. In this context, cellular membrane protrusions and the actin cytoskeleton are essential to guide DC migration towards chemotactic signals. Caveolin-1 (CAV1) is a scaffolding protein that modulates signaling pathways leading to remodeling of the actin cytoskeleton and enhanced migration of cancer cells. However, whether CAV1 is relevant for DC function and specifically for DC migration to LNs is unknown. Here, we show that CAV1 expression is upregulated in DCs upon LPS- and TNF-α-induced maturation. CAV1 deficiency did not affect differentiation, maturation, or the ability of DCs to activate CD8+ T cells in vitro. However, CAV1-deficient (CAV1-/-) DCs displayed reduced in vivo trafficking to draining LNs in control and inflammatory conditions. In vitro, CAV1-/- DCs showed reduced directional migration in CCL21 gradients in transwell assays without affecting migration velocity in confined microchannels or three-dimensional collagen matrices. In addition, CAV1-/- DCs displayed reduced activation of the small GTPase Rac1, a regulator of actin cytoskeletal remodeling, and lower numbers of F-actin-forming protrusions. Furthermore, mice adoptively transferred with peptide-pulsed CAV1-/- DCs showed reduced CD8+ T cell responses and antitumor protection. Our results suggest that CAV1 promotes the activation of Rac1 and the formation of membrane protrusions that favor DC chemotactic trafficking toward LNs where they can initiate cytotoxic T cell responses.
Forcing Entry into the Nucleus.
AlexisLomakin, GuilhermeNader, MatthieuPiel
Cell - 43 547-548 - https://doi.org/10.1016/j.devcel.2017.11.015 - 2017
Nuclear pore complexes tightly regulate nucleo-cytoplasmic transport, controlling the nuclear concentration of several transcription factors. In a recent issue of Cell, Elosegui-Artola et al. (2017) show that nuclear deformation modulates the nuclear entry rates of YAP/TAZ via nuclear pore stretching, clarifying how forces affect gene transcription.
ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X receptors.
Sáez PJ, Vargas P, Shoji KF, Harcha PA, Lennon-Duménil AM, Sáez JC
Sci Signal. - 10(506) 7107 - doi: 10.1126/scisignal.aah7107. - 2017
Upon its release from injured cells, such as infected, transformed, inflamed, or necrotic cells, extracellular adenosine-5'-triphosphate (ATP) acts as a danger signal that recruits phagocytes, such as neutrophils, macrophages, and dendritic cells (DCs), to the site of injury. The sensing of extracellular ATP occurs through purinergic (P2) receptors. We investigated the cellular mechanisms linking purinergic signaling to DC motility. We found that ATP stimulated fast DC motility through an autocrine signaling loop, which was initiated by the activation of P2X7 receptors and further amplified by pannexin 1 (Panx1) channels. Upon stimulation of the P2X7 receptor by ATP, Panx1 contributed to fast DC motility by increasing the permeability of the plasma membrane, which resulted in supplementary ATP release. In the absence of Panx1, DCs failed to increase their speed of migration in response to ATP, despite exhibiting a normal P2X7 receptor-mediated Ca2+ response. In addition to DC migration, Panx1 channel- and P2X7 receptor-dependent signaling was further required to stimulate the reorganization of the actin cytoskeleton. In vivo, functional Panx1 channels were required for the homing of DCs to lymph nodes, although they were dispensable for DC maturation. These data suggest that P2X7 receptors and Panx1 channels are crucial players in the regulation of DC migration to endogenous danger signals.

396 publications.