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Digital Microfluidic Isolation of Single Cells for -Omics

ABSTRACT: Proteomic data from 0,1, and 5 U87 cells.A custom single cell proteomic pipeline was developed to collect cell lysates and evaluate them by LC-MS/MS.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jiaxi Peng

LAB HEAD: Aaron R. Wheeler

PROVIDER: PXD019958 | Pride | 2020-10-26

REPOSITORIES: Pride

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			Action	DRS
	0cell191115_2.raw	Raw
	0cell191115_2txt.rar	Other
	0cell191115_3.raw	Raw
	0cell191115_3txt.rar	Other
	0cell191115_5.raw	Raw

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Publications

Digital microfluidic isolation of single cells for -Omics.

Lamanna Julian J Scott Erica Y EY Edwards Harrison S HS Chamberlain M Dean MD Dryden Michael D M MDM Peng Jiaxi J Mair Barbara B Lee Adam A Chan Calvin C Sklavounos Alexandros A AA Heffernan Austin A Abbas Farhana F Lam Charis C Olson Maxwell E ME Moffat Jason J Wheeler Aaron R AR

Nature communications 20201111 1

We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell ...[more]

PMID: 33177493

Similar Datasets

Digital microfluidic isolation of single cells for -Omics.

Project description:We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.

| S-EPMC7658233 | biostudies-literature

Digital microfluidic immunocytochemistry in single cells.

Project description:We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations-for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population.

| S-EPMC4491823 | biostudies-literature

A digital microfluidic system with 3D microstructures for single-cell culture.

Project description:Despite the precise controllability of droplet samples in digital microfluidic (DMF) systems, their capability in isolating single cells for long-time culture is still limited: typically, only a few cells can be captured on an electrode. Although fabricating small-sized hydrophilic micropatches on an electrode aids single-cell capture, the actuation voltage for droplet transportation has to be significantly raised, resulting in a shorter lifetime for the DMF chip and a larger risk of damaging the cells. In this work, a DMF system with 3D microstructures engineered on-chip is proposed to form semi-closed micro-wells for efficient single-cell isolation and long-time culture. Our optimum results showed that approximately 20% of the micro-wells over a 30 × 30 array were occupied by isolated single cells. In addition, low-evaporation-temperature oil and surfactant aided the system in achieving a low droplet actuation voltage of 36V, which was 4 times lower than the typical 150 V, minimizing the potential damage to the cells in the droplets and to the DMF chip. To exemplify the technological advances, drug sensitivity tests were run in our DMF system to investigate the cell response of breast cancer cells (MDA-MB-231) and breast normal cells (MCF-10A) to a widely used chemotherapeutic drug, Cisplatin (Cis). The results on-chip were consistent with those screened in conventional 96-well plates. This novel, simple and robust single-cell trapping method has great potential in biological research at the single cell level.

| S-EPMC8433300 | biostudies-literature

Integrated Single-Cell Proteomics Analysis on an Active Matrix Digital Microfluidic Chip

Project description:We introduce and implement a comprehensive single-cell proteomics sample pre-treatment solution based on an active matrix digital microfluidics chip(AM-DMF-SCP). This platform accommodates high-throughput single-cell isolation and seamless, non-destructive sample pre-treatment, characterized by its efficiency, speed, stability, and ease of use, thereby overcoming the longstanding bottleneck in single-cell protein sample preparation.

2024-04-15 | PXD048434 |

Genome-level Cell Responses to Digital Microfluidic Manipulation

Project description:Analysis of mammalian cell responses to digital microfluidic manipulation. BA/F3 cells were manipulated on devices with 15 min continuous actuation at 400Vpp at 1 kHz or 18 kHz and compared to untreated control cells and cells heat shocked at 42C, 47C or 52C. Total RNA isolated from Ba/F3 murine cells, untreated or exposed to digital microfludic manipulation or heat shock.

2013-05-01 | E-GEOD-43507 | biostudies-arrayexpress

Genome-level Cell Responses to Digital Microfluidic Manipulation

2013-05-01 | GSE43507 | GEO

Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR.

Project description:Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve 'quantitative' measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10(-6)) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1×10(-7) or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur.

| S-EPMC3763562 | biostudies-literature

Regulation of gene expressions in vivo by anti-VEGF therapy [Mouse430_2]

Project description:U87-EV human glioblastoma xenograft tumours is therapeutically treated by bevacizumab, a humanized anti-human VEGF mAb, when tumour is established in BALB/c SCID mice. At the end point, collect tumour samples and extracted total RNA for microarray to investigate the gene profile changes compared to control. These include the genes from human tumour cells and mouse host stroma cells.

2014-11-09 | GSE39221 | GEO

Regulation of gene expressions in vivo by anti-VEGF therapy

2014-04-25 | GSE37956 | GEO

A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection.

Project description:In this work, a droplet microfluidic platform for genetic mutation detection from crude biosample is described. Single-stream integration of nucleic acid isolation and amplification is realized on a simple fluidic cartridge. Subsequent DNA melting curve is employed with signal normalizing algorithm to differentiate heterozygous K-ras codon 12 c.25G>A mutant from the wildtype. This technique showcases an alternative to modular bench-top approaches for genetic mutation screening, which is of interest to decentralized diagnostic platforms.

| S-EPMC4223799 | biostudies-literature

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