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Targeting Cells The answer to Cancer Vasculature for you to Induce Cancer Infarction.
Using this touch prep-qSMLM methodology, we interrogated the density and nano-organization of human epidermal growth factor receptor 2 (HER2) in fresh breast cancer tissues. Touch prep-qSMLM agreed well with current clinical methods. Importantly, touch prep-qSMLM can be easily extended to other pathological conditions and ultimately used in precision medicine.Droplette (US 9,700,686 B2 and PCT/US2016/035695) is the first portable and contact-free transdermal technology comprising the unique combination of a piezoelectric transducer and a pneumatic diaphragm pump to deliver large biomolecules including nucleic acid therapeutics (NATs) deep into cells, and into skin and soft tissue for effective delivery over short timescales. BAPTA-AM The droplets that come out of the device are 10-50× smaller upon impact than what is created through other commercial atomizers, such as the piezoelectric transducer alone. This device has been tested extensively in vitro, in vivo and in IRB approved human studies. The Droplette device delivers metered doses using a water droplet dispersal technology already commonly used in humidifier devices, by utilizing a piezoelectric material. Three key innovations make this device technically novel and tailored specifically for both field and lab use (1) The combination of the piezo and pump to generate sub-micron drug-loaded droplets that penetrate celhe Droplette system without the need for traditional transfection reagents or methods.Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations. MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles with sub-micrometer size that are released by various cells. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Growing evidence has shown important biological roles and clinical relevance of EVs in diseases. For EV-related studies to thrive, rapid efficient isolation of pure EVs is a prerequisite. However, lengthy procedure, low yield, low throughput, and high contaminants stemmed from existing isolation approaches hamper both basic research and large-scale clinical implementation. We have shown that lipid nanoprobes (LNP) enable spontaneous labeling and rapid isolation of EVs by coupling with magnetic enrichment. Recently, we further developed a one-step EV isolation platform that utilizes EV size-matched silica nanostructures and surface-conjugated LNPs with an integrated microfluidic mixer. EVs, derived from up to 2-ml clinical plasma, can be processed with this point-of-care device using optimized flow rate. Subsequently, contents of isolated EVs can be extracted on-chip and eluted from the device for downstream molecular analyses. The LNP-functionalized microfluidic device combined with state-of-the-art analysis platforms could have great potential in promoting EV-centered research and clinical use in the future.Node-Pore Sensing, NPS, is an extremely versatile and powerful technique for the analysis of cells and the detection of extracellular vesicles (EVs). NPS involves measuring the modulated current pulse caused by a cell transiting a microfluidic channel that has been segmented by a series of inserted nodes. As the current pulse reflects the number of nodes and segments of the channel, NPS can achieve exquisite sensitivity. Thus, when used as a Coulter counter, NPS can measure the sub-micron size increase of antibody-coated colloids to which EVs are specifically bound. By simply inserting between two nodes a "contraction" channel through which cells can squeeze, one can mechanically phenotype cells. We discuss the details of performing these two NPS applications.Changes in intracellular GTP levels, even incremental ones, profoundly affect the activity of several GTP-binding proteins ultimately resulting in alteration of several basal cellular phenotypes including cell motility, invasion, and tumorigenesis. However, until recently, no tools were available for GTP quantification in live cells. Therefore, in the current chapter, we describe the methodology for the quantitative assessment of spatiotemporal changes in GTP levels in the cells using genetically encoded fluorescent ratiometric GTP sensors termed GEVALs for GTP evaluators.Posttranslational modification (PTM) enzymes are important modulators of protein structure and function. They typically act by chemically modifying amino acids, often on side chain functional groups, to change the physiochemical landscape of the protein and thus its biophysical behavior. In particular, protein kinases are enzymes that transfer phosphate from ATP to serine, threonine, or tyrosine in protein substrates. They are key regulators of vital cellular pathways such as survival, proliferation, and apoptosis, and their dysregulation in the context of cancer has been widely investigated for the purpose of development of anticancer drugs. However, several critical questions pertaining to their physiology, such as heterogeneity of kinase signaling within and between cells, and other factors that may play into the mechanisms of drug resistance, remain unanswered. Many of the current strategies to measure kinase activity lack the scope, subcellular resolution, and real-time monitoring ability needed to obtaiange occurs, this approach is compatible with other PTMs (such as acetylation, methylation), and so the considerations for kinase FLIM probe design described in this chapter should be broadly applicable for other PTMs as well.The ability to track and isolate unique cell lineages from large heterogeneous populations increases the resolution at which cellular processes can be understood under normal and pathogenic states beyond snapshots obtained from single-cell RNA sequencing (scRNA-seq). Here, we describe the Control of Lineages by Barcode Enabled Recombinant Transcription (COLBERT) method in which unique single guide RNA (sgRNA) barcodes are used as functional tags to identify and recall specific lineages of interest. An sgRNA barcode is stably integrated and actively transcribed, such that all cellular progeny will contain the parental barcode and produce a functional sgRNA. The sgRNA barcode has all the benefits of a DNA barcode and added functionalities. Once a barcode pertaining to a lineage of interest is identified, the lineage of interest can be isolated using an activator variant of Cas9 (such as dCas9-VPR) and a barcode-matched sequence upstream of a fluorescent reporter gene. CRISPR activation of the fluorescent reporter will only occur in cells producing the matched sgRNA barcode, allowing precise identification and isolation of lineages of interest from heterogeneous populations.Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.There is growing interest in breaking down tissues into the individual cellular constituents so that those cells can be identified, assayed for functional characteristics, or utilized for therapeutic purposes. A major driver is the development of single cell analysis methods, which are best poised to assess cellular heterogeneity and discover rare cells. Current tissue dissociation methods are inefficient, produce variable results, and require many labor-intensive, time-consuming steps. To address these shortcomings, we have developed three different microfluidic technologies to perform the critical steps of tissue digestion, disaggregation, and filtration with improved dissociation efficiency and speed. These devices will make it possible to process tissue into single cells for various downstream applications in a rapid and automated fashion.Here we present a protocol for interrogating AKT signaling activities in living single cells, using a pair of cyclic peptide-based fluorescent probes. These probes are encapsulated in liposomes and delivered into cells, where they continuously report on AKT signaling activities through a Föster resonance energy transfer mechanism. We describe the use of a microwell chip to achieve single-cell resolution and demonstrate the procedure for on-chip immunostaining. Finally, we provide a method for data extraction, correction, and processing.The interaction between cells and their surrounding microenvironment has a crucial role in determining cell fate. In many pathological conditions, the microenvironment drives disease progression as well as therapeutic resistance. A number of challenges arise for researchers examining these cell-microenvironment interactions (1) Tissue microenvironments are combinatorial and dynamic systems, and in pathological situations like cancer, microenvironments become infamously chaotic and highly heterogeneous. (2) Cells exhibit heterogeneous phenotypes, and even rare cell subpopulations can have a substantial role in tissue homeostasis and disease progression. This chapter discusses technical aspects relevant to dissecting cell-microenvironment interaction using the Microenvironment Microarray (MEMA) platform, which is a cell-based functional high-throughput screening of interactions between cells and combinatorial microenvironments at the single-cell level. MEMA provides insights into how cell phenotype and function is elicited by microenvironmental components.
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