Tobey-microfluidics
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB48713 | ENA |
REPOSITORIES: ENA
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Project description:Neutrophils play critical roles in modulating the immune response. However, neutrophils have a short circulating half life, are readily stimulated in vitro, and have low levels of cellular mRNA when compared to other blood leukocyte populations. All of these factors have made it difficult to evaluate neutrophils from clinical populations for molecular and functional studies. Here we present a robust methodology for rapidly isolating neutrophils directly from whole blood and develop âon- chipâ processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and demonstrate the ability of the device to discriminate subtle differences in the genomic and proteomic response of peripheral blood neutrophils to direct and indirect stimulation. Lastly, we implement this tool as part of a near patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury and demonstrate that this technique is easy to use by nurses and technical staff yielding excellent quality and sufficient quantity of mRNA for sensitive genomic readout of the host response to injury 1. Ex vivo Stimulation Studies: We assessed whether the relatively small number of isolated neutrophils captured by the microfluidics cassettes would impact the resulting genomic sensitivity and potential discriminatory genomic capabilities in response to various stimuli. To do this, we compared the genome-wide expression profile in neutrophils from 4 independent repeated experiements under three conditions - untimulated, ex vivo activation with either Escherichia coli lipopolysaccharide (LPS), or with granulocyte-macrophage colony-simulating factor (GM-CSF) and interferon-gamma (INF-g) (referred to as GM+I). In both protocols, whole blood was stimulated ex vivo24 to allow leukocyte and plasma protein interactions. 2. Inter-subject Reproducibility: To further ensure the reliability of the of the microfluidics cassette isolation method, we directly compared the gene expression of neutrophils captured in the microfluidics cassettes with neutrophils isolated using density centrifugation with Ficoll-dextran. We performed parallel neutrophil isolations using both methodologies from five different healthy volunteers and processed the cell lysates for microarray analysis using identical protocols.
2010-06-16 | E-GEOD-22103 | biostudies-arrayexpress
Project description:The fungal toxin-encoding genes are highly upregulated in the vegetative mycelium upon challenge with the predator. Our recent studies in microfluidics have shown that latter induction is spatially restricted to parts of the vegetative mycelium that is in direct contact with the predator. In order to dissect the defensome of a multicellular fungus against a predator, here, we performed RNA - sequencing of mushroom Coprinopsis cinerea upon challenged with fungivorous nematode Aphelenchus avenae in Microfluidics device at three different time points. We analyzed hyphae that were collected from a microfluidics device where they have been in direct contact with or cultivated without A. avenae.
2018-11-30 | E-MTAB-7005 | biostudies-arrayexpress
Project description:Methylomic studies require substantial amounts of DNA samples and this restriction hinders applications involving scarce animal or patient samples with direct biomedical relevance. Here we report a microfluidics-based reduced representative bisulfite sequencing protocol, MIcrofluidic Diffusion-based RRBS (MID-RRBS), that permits methylomic profiling with sub-1 ng starting DNA. Using this technology, we studied DNA methylation in NeuN+ and NeuN- fractions isolated from mouse cerebellum, revealing cell-type specific methylomic patterns. We also studied the DNA methylation in NeuN+ nuclei isolated from clozapine or vehicle treated mouse frontal cortex.
2018-02-15 | GSE88716 | GEO
Project description:A microfluidics technology was implemented to the immunoaffinity purification process of MHC peptides in Ligandomics/Immunopeptidomics. The thus purified HLA peptides were analysed by LCMS with the nanoElute LC and TimsTOF Pro Mass Spectrometer from Bruker. The aim of the microfluidics implementation was to improve the sensitivity and robustness while also reducing antibody and other material requirements in the immunoaffinity purification protocol.
2022-02-17 | PXD022194 | Pride
Project description:The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.
2015-06-05 | GSE62050 | GEO
Project description:The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells. In this experiment we mixed 2 cell types (mES mEF) and then using single cell novel approach we could be able to find each cell (using its barcode) and assign it to mES of mEF and to produce mES and mEF aggregate bam files (converted to bed for GEO submission). 1152_RNA_RTprimers_Barcodes.txt: A list of all 1152 barcodes sequenced for Read2 fastq files.
2015-06-05 | E-GEOD-62050 | biostudies-arrayexpress
Project description:We have developed a microfluidics-based in vitro model of the human gut allowing co-culture of human and microbial cells and subsequent multi-omic assessment of the effect of the co-culture on the host transcriptome. We compare the transcriptional changes induced in the human epithelial cell line, Caco-2 after co-culture with Lactobacillus rhamnosus GG or a consortium of Lactobacillus rhamnosus GG and Bacteroides caccae.
2016-05-05 | GSE79383 | GEO
Project description:Despite the physiological and pathophysiological significance of microenvironmental gradients, tools for generating such gradients and analysing their impact on cellular phenotypes are lacking. Here we present an integrated microfluidics-based workflow for mimicking extracellular pH gradients characteristic of solid tumors, and studying their multifactorial impact on cancer cells. Our microfluidics device generates a pH gradient across cancer cell 3D cultures in an extracellular matrix. The gradient, validated using pH-sensitive fluorophores can be rapidly controlled to represent spatiotemporal microenvironmental changes, and the device allows high resolution live imaging of, e.g., cell motility and chemotaxis. The device can be reopened, allowing immunofluorescence analysis of phenotypes and spatially resolved analysis of gene expression changes across the pH gradient. The workflow is easily adaptable for other gradients and multiple cell types, making it broadly applicable for integrated analysis of roles of microenvironmental gradients in biology.
2024-05-07 | GSE242233 | GEO
Project description:Mass spectrometry (MS)-based immunopeptidomics is an attractive antigen discovery method with growing clinical implications, especially for personalized cancer immunotherapies. However, the current experimental approach to extract HLA-I-restricted peptides requires a bulky sample source, which remains a challenge for obtaining clinical tumor samples. We present an innovative streamlined workflow that requires a low sample volume. The workflow integrates the immunoaffinity purification (IP) and C18 peptide cleanup steps on a single microfluidics platform with automated liquid handling and minimal sample transfers, resulting in higher assay sensitivity. We also demonstrate how state-of-the-art data-independent acquisition (DIA) MS method further enhances the depth and reproducibility of tandem mass spectrometry (MS2) spectra-based peptide sequencing. As a result, over 4,000 and 5,000 HLA-I restricted peptides can be reliably identified from as low as 0.2 million human RA957 cells and a melanoma tissue of merely 5 mg, respectively. We also identified multiple known immunogenic tumor-associated antigens and hundreds of peptides derived from non-canonical protein sources. The presented workflow is a powerful tool for identifying the immunopeptidome of sparse samples.
2023-05-12 | PXD040858 | Pride
Project description:Combined community health programs aiming at health education, preventive antiparasitic chemotherapy, and vaccination of pigs have proven their potential to regionally reduce and even eliminate Taenia solium infections that are associated with a high risk of neurological disease through ingestion of T. solium eggs. Yet it remains challenging to target T. solium endemic regions precisely or to make exact diagnoses in individual patients. One major reason is that the widely available stool microscopy may identify Taenia ssp. eggs in stool samples as such, but fails to distinguish between invasive (T. solium) and less invasive Taenia (T. saginata, T. asiatica, and T. hydatigena) species. The identification of Taenia ssp. eggs in routine stool samples often prompts a time-consuming and frequently unsuccessful epidemiologic workup in remote villages far away from a diagnostic laboratory. Here we present “mail order” single egg RNA-sequencing, a new method allowing the identification of the exact Taenia ssp. based on a few eggs found in routine diagnostic stool samples. We provide first T. solium transcriptome data, which show extremely high mitochondrial DNA (mtDNA) transcript counts that can be used for subspecies identification. “Mail order” RNA-sequencing can be administered by health personnel equipped with basic laboratory tools such as a microscope, a Bunsen burner, and access to an international post office for shipment of samples to a next generation sequencing facility. Our suggested workflow combines traditional stool microscopy, RNA-extraction from single Taenia eggs with mitochondrial RNA-sequencing, followed by bioinformatic processing with a basic laptop computer. The workflow could help to better target preventive healthcare measures and improve diagnostic specificity in individual patients based on incidental findings of Taenia ssp. eggs in diagnostic laboratories with limited resources.
2021-11-06 | GSE175668 | GEO