Spatial Marking and Quantitative Counting Cell Membrane αIIbβ3Integrin in Single Cells via Peptide-Au Cluster Probes

2015 ◽  
Vol 1802 ◽  
pp. 25-31
Author(s):  
Lina Zhao ◽  
Xueyun Gao
Keyword(s):  
Cell Membrane ◽  
Single Cell ◽  
Disease Progression ◽  
Drug Response ◽  
Single Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Au Cluster ◽  
Tight Correlation ◽  
Counting Cell

ABSTRACTWe propose a theoretical designed peptide-Au cluster probe and realize spatially marking and quantitatively counting αIIbβ3integrin via this novel nanoprobe. On single cell level, we find the number of αIIbβ3integrin ranges from 5.75 to 9.11×10-17mol for the heteroexpression of individual cells. Because the variation of proteins (including integrin and other biomarkers) on single cell has a tight correlation with many serious human diseases, our quantifying protein method on a single cell level is helpful for estimating the disease progression for diagnosis and detecting the drug response for therapy.

scholarly journals Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 285
Author(s):  
Eszter Széles ◽  
Krisztina Nagy ◽  
Ágnes Ábrahám ◽  
Sándor Kovács ◽  
Anna Podmaniczki ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Single Cell ◽  
Single Cells ◽  
Fluorescence Induction ◽  
Photochemical Quenching ◽  
Support Surface ◽  
Single Cell Level ◽  
Microfluidic Platforms ◽  
Cell Level ◽  
Non Photochemical Quenching

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

scholarly journals Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

2009 ◽  
Vol 75 (13) ◽  
pp. 4550-4556 ◽  
Author(s):  
Vicky G. Kastbjerg ◽  
Dennis S. Nielsen ◽  
Nils Arneborg ◽  
Lone Gram
Keyword(s):  
Listeria Monocytogenes ◽  
Single Cell ◽  
Intracellular Ph ◽  
Bacterial Population ◽  
Single Cells ◽  
Viable Cell ◽  
Population Subdivision ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Counts

ABSTRACT Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

scholarly journals Combined Molecular Genetic and Cytogenetic Analysis from Single Cells after Isothermal Whole-Genome Amplification

2011 ◽  
Vol 57 (7) ◽  
pp. 1032-1041 ◽  
Author(s):  
Thomas Kroneis ◽  
Jochen B Geigl ◽  
Amin El-Heliebi ◽  
Martina Auer ◽  
Peter Ulz ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Molecular Genetic ◽  
Target Cells ◽  
Whole Genome ◽  
Single Cell Level ◽  
Cell Level ◽  
Genome Amplification

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

scholarly journals Single-Cell Analysis of the Transcriptional Response in AML Patients Treated with BET-Inhibitors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5120-5120
Author(s):  
Sophia Miliara ◽  
Bogumil Kaczkowski ◽  
Takahiro Suzuki ◽  
Huthayfa Mujahed ◽  
Maasaki Furuno ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Drug Response ◽  
Transcriptional Profiling ◽  
Single Cell Level ◽  
Cell Heterogeneity ◽  
Cell Level ◽  
Bet Inhibitors ◽  
Single Cell Profiling

Abstract Acute Myeloid Leukemia (AML) is the most common myeloid leukemia in adults. Although substantial progress has been made in recent years, the long-term prognosis for patients remains poor which is mainly due to the dated treatments that consist of cytotoxic drugs with low specificity. AML is a clonal disease with multiple co-existing clones in each patient. Often, patients that initially respond to treatment may develop resistance due to lingering leukemic stem cells (LSC), or sub-clones that survive the treatment and cause a relapse. Therefore, novel therapeutic strategies are needed to fully eradicate all leukemic cells. AML has a strong epigenetic component meaning mutations in genes encoding epigenetic regulators are frequently acquired during early AML development, and are present in the initiating clones. Thus, targeting the epigenetic machinery may offer a new avenue for AML treatment. Among the newer epigenetic drugs are BET inhibitors, which bind reversibly to bromodomains of BRD proteins and prevent protein-protein interactions with acetylated histones and transcriptions factors. One of the most promising BET inhibitors is OTX015, which has already been in Phase II clinical trials for AML in the U.S. (Braun & Gardin, Expert Opinion on Investigational Drugs, 2017). We aim to analyze the heterogeneous response to OTX015 in AML, and normal stem/progenitor, cells in order to dissect the BET-inhibitor response. The main focus is the specific transcriptional signatures at promoters and enhancers as enhancers, and especially super-enhancers, have previously been shown to be sensitive to BET-inhibitors (Loven et al, Cell, 2013). To this effect, we have established a protocol that allowed for the transcriptional profiling of single cells from AML patients that were at different differentiation stages, using FACS- sorting. The patients were obtained from the Swedish Acute Leukemia Registry. To decrease population heterogeneity, the project focused on distinct subgroups of AML that previously has been shown to be sensitive for BET inhibitors. The different isolated AML, and normal progenitor populations, were exposed to OTX015 for 48hrs, and processed with both bulk transcriptional profiling of the general cell population response, and single cell profiling to analyze cell heterogeneity, and single cell response. For the transcriptional profiling, we utilized a unique technique called Cap Analysis of Gene Expression (CAGE), a powerful 5' start profiling technology, that allows for the identification of the transcription start site at base pair resolution, and determination of enhancer activity based on enhancer RNA expression. The single cell profiling was performed using C1 CAGE, which is a single-cell implementation CAGE (Kouno et al, bioRxiv 330845, 2018).We envision that the heterogenic transcriptional drug response, on the single-cell level, in AML and normal stem/progenitor cells will lead to the identification of key genes and pathways involved in the differential drug response. Additionally, the application of CAGE technology will lead to discovery of specific transcriptional signatures at promoters and enhancers that may be predictive of drug resistance. Clinical significance: Leukemic cell heterogeneity remains the main problem in AML, as chemotherapy often fails to completely eradicate all AML sub-clones including LSC, leading to relapses and high mortality of the disease. This study will shed light to the unique features of AML cell heterogeneity and how their drug response differs, not only between AML cells, but also between AML cells and their normal counterparts, on the single-cell level, based on the response to OTX015. The significance will be two-fold: the in-depth characterization of the features in AML populations and normal cells, and the potential this study will provide for novel, more targeted, combination treatments in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Generation of genetic tools for gauging multiple gene expression at single cell level

2021 ◽  
Author(s):  
Marta Mellini ◽  
Massimiliano Lucidi ◽  
Francesco Imperi ◽  
Paolo Visca ◽  
Livia Leoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Image Processing ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Phenotypic Heterogeneity ◽  
Single Cell Level ◽  
Cell Level ◽  
Multiple Gene ◽  
Genetic Tools

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple gene expression at the single cell level has limited the understanding of phenotypic heterogeneity. In order to investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple gene expression at the single cell level has been generated. This tool, named pRGC, consists in a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP) and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population, without the need of image-processing for spectral crosstalk correction. In addition, two pRGC variants have been generated for either i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome, that is suitable for long-term experiments in the absence of antibiotic selection, or ii) replication in bacterial genera other than Pseudomonas. The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity. IMPORTANCE Within a bacterial population single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial sub-populations with distinct phenotypes. The analysis of gene expression at the single cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple gene expression at the single cell level by fluorescent microscopy, without the need of advanced image-processing procedures. A proof of concept has been provided by investigating iron-uptake and iron-storage gene expression in response to iron availability in P. aeruginosa.

A Microfabricated Chip for the Study of Cell Electroporation

2000 ◽  
Author(s):  
Yong Huang ◽  
Boris Rubinsky
Keyword(s):  
Silicon Nitride ◽  
Cell Membrane ◽  
Single Cell ◽  
Electrical Potential ◽  
Single Cell Level ◽  
Cell Level ◽  
Electrical Potentials ◽  
Silicon Nitride Membrane ◽  
Micro Hole ◽  
Flow Through

Abstract It has been observed that when certain electrical potentials are applied across a cell they can induce the formation of pores in the cell membrane and consequently increase the permeability of the cell to macromolecules. This phenomenon is known as electroporation. Since the first report on gene transfer by electroporation1, it has become a standard method for introduction of macromolecules into cells2 3 4. Currently, electroporation is normally done in batches of cells between electrodes and there is little control over the permeabilization of individual cells. Therefore, it is very difficult to study the fundamental biophysics of cell membrane electro-permeabilization, which is not yet understood, and to design optimal and reversible electroporation protocols for individual cells2 3. Although the biophysics of electroporation are still not fully understood, indirect evidence shows that micro aqueous pores with diameters of tens to hundreds of angstroms are created in cell membrane due to the electrical field induced structural rearrangement of the lipid bilayer5. It occurred to us that if electroporation induces pores in the cell membrane than, in a state of electroporation, a measurable current should flow through the individual cell. From this idea, we have developed a new micro-electroporation technology that employs a “bionic” chip to study and control the electroporation process in individual cells. The micro-electroporation chips are designed and fabricated using standard silicon microfabrication technology. Figure 1 shows the schematic of the chip in cross section. Each chip is a three-layer device that consists of two translucent poly silicon electrodes and a silicon nitride membrane, which all together form two fluid chambers. The two chambers are interconnected only through a micro hole on the dielectric silicon nitride membrane. In a typical process, the two chambers are filled with conductive solutions and one chamber contains biological cells. Individual cells can be captured in the micro hole and thus incorporated in the electrical circuit between the two electrodes of the chip. When the cell is in its normal state no current flows through the insulating lipid bilayer and consequently between the electrodes. However, when the electrical potential across the electrodes is sufficient to induce electroporation, a measurable current will flow through the pores of the cell membrane and between the electrodes. Measuring currents through the bionic chip as a function of electrical potential will determine the potential that induces the electroporation. The chip behaves somewhat similarly to an electrical diode, with no current at potentials that do not induce electroporation and currents at potentials that induce electroporation. With the ability to manipulate individual cells and detect the electrical potentials that induce electroporation in each cell, the chip can be used to study the fundamental biophysics of membrane electro-permeabilization on single cell level and in biotechnology, for controlled introduction of macromolecules, such as gene constructs, into individual cells. We anticipate that this new technology will change the way in which electroporation is done and will provide key understanding of the biophysical processes that lead to cell electroporation. In this paper, first the design, fabrication process and modeling of the microelectroporation chip are described in details. Subsequently, experiment methods and results are presented and discussed, demonstrating the feasibility of altering cell membrane permeability and facilitating intercellular mass transfer in a more controlled way on single cell level. Finally, the potential applications of the micro-electroporation chips and future research directions are discussed. Figure 2 demonstrates how cell membrane electroporation can be investigated through monitoring and analyzing chip current-voltage signatures.

scholarly journals A method of quantifying centrosomes at the single-cell level in human normal and cancer tissue

2019 ◽  
Vol 30 (7) ◽  
pp. 811-819 ◽  
Author(s):  
Mengdie Wang ◽  
Beatrice S. Knudsen ◽  
Raymond B. Nagle ◽  
Gregory C. Rogers ◽  
Anne E. Cress
Keyword(s):  
Single Cell ◽  
Human Tissue ◽  
Single Cells ◽  
Cancer Tissue ◽  
Single Cell Level ◽  
Cell Level ◽  
Tissue Samples ◽  
Tumor Subtypes ◽  
Different Types ◽  
Pericentriolar Material

Centrosome abnormalities are emerging hallmarks of cancer. The overproduction of centrosomes (known as centrosome amplification) has been reported in a variety of cancers and is currently being explored as a promising target for therapy. However, to understand different types of centrosome abnormalities and their impact on centrosome function during tumor progression, as well as to identify tumor subtypes that would respond to the targeting of a centrosome abnormality, a reliable method for accurately quantifying centrosomes in human tissue samples is needed. Here, we established a method of quantifying centrosomes at a single-cell level in different types of human tissue samples. We tested multiple anti-centriole and pericentriolar-material antibodies to identify bona fide centrosomes and multiplexed these with cell border markers to identify individual cells within the tissue. High-resolution microscopy was used to generate multiple Z-section images, allowing us to acquire whole cell volumes in which to scan for centrosomes. The normal cells within the tissue serve as internal positive controls. Our method provides a simple, accurate way to distinguish alterations in centrosome numbers at the level of single cells.

A quantitative analysis of testosterone action on FSH secretion from individual pituitary cells using the cell immunoblot assay

1996 ◽  
Vol 148 (3) ◽  
pp. 427-433 ◽  
Author(s):  
K Noguchi ◽  
J Arita ◽  
A Nagamoto ◽  
M Hosaka ◽  
F Kimura
Keyword(s):  
Single Cell ◽  
Anterior Pituitary ◽  
Single Cells ◽  
Male Rat ◽  
Image Analyzer ◽  
Pituitary Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Anterior Pituitary Cells

Abstract We investigated the effects of testosterone on FSH secretion from male rat anterior pituitary cells in culture at the single cell level. Anterior pituitary cells cultured with or without 10 ng/ml testosterone for 72 h were mono-dispersed and subjected to cell immunoblot assays for FSH. Cell blots specific for FSH were quantified by means of a microscopic image analyzer. The number of FSH-secreting cells detected as immunoreactive cell blots on the transfer membrane represented 4·1% of total pituitary cells applied on the membrane. The amount of FSH secreted by single cells varied from <20 to >8 000 fg/cell/h. The number of FSH-secreting cells was not changed by the addition of 10 ng/ml testosterone into the culture medium. Testosterone administration increased the mean FSH secretion by 64% after 3 h incubation, resulting in a shift to the right in the frequency distribution of FSH secretion from single cells. The total amount of FSH, namely the sum of FSH secreted by each FSH-secreting cell, was increased by 92% by the addition of testosterone. However, mean amounts of FSH secretion by the top ten cells of the largest secretor subgroup (>5 pg/cell/3 h) were not different between control and testosterone-treated groups. The present study analyzed, for the first time, FSH secretion from rat anterior pituitary cells at the single cell level. The results suggest that stimulation by testosterone of FSH secretion in vitro is not due to an increase in the number of FSH-secreting cells but to an increase in FSH secretion from each cell. Journal of Endocrinology (1996) 148, 427–433

scholarly journals Efficient analysis of a small number of cancer cells at the single-cell level using an electroactive double-well array

Lab on a Chip ◽  
2016 ◽  
Vol 16 (13) ◽  
pp. 2440-2449 ◽  
Author(s):  
Soo Hyeon Kim ◽  
Teruo Fujii
Keyword(s):  
Single Cell ◽  
Cancer Cells ◽  
Single Cells ◽  
Capture Efficiency ◽  
Single Cell Level ◽  
Cell Capture ◽  
Cell Level ◽  
Cell Trapping ◽  
Highly Efficient ◽  
Single Cell Trapping

The electroactive double well-array consists of trap-wells for highly efficient single-cell trapping using dielectrophoresis (cell capture efficiency of 96 ± 3%) and reaction-wells that confine cell lysates for analysis of intracellular materials from single cells.

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