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.

scholarly journals Centrifugal Microfluidics Traps for Parallel Isolation and Imaging of Single Cells

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 149 ◽  
Author(s):  
Adam Snider ◽  
Ileana Pirozzi ◽  
Anubhav Tripathi
Keyword(s):  
Single Cell ◽  
Cancer Cells ◽  
Single Cells ◽  
Unmet Need ◽  
Centrifugal Microfluidics ◽  
Cancer Tissue ◽  
Single Cell Level ◽  
Cell Level ◽  
Tissue Samples ◽  
Potential Applications

Analysis at the single cell level has becoming an increasingly important procedure to diagnose cancer tissue biopsies. These tissue samples are often heterogeneous and consist of 1000–15,000 cells. We study the use of centrifugal microfluidics to isolate single cells into micro chambers. We describe the optimization of our microfluidics flow device, characterize its performance using both polystyrene beads as a cell analogue and MCF-7 breast cancer cells, and discuss potential applications for the device. Our results show rapid isolation of ~2000 single cell aliquots in ~20 min. We were able to occupy 65% of available chambers with singly occupied cancer cells, and observed capture efficiencies as high as 80% using input samples ranging from 2000 to 15,000 cells in 20 min. We believe our device is a valuable research tool that addresses the unmet need for massively parallel single cell level analysis of cell populations.

scholarly journals A step towards enzyme-free tissue dissociation

2019 ◽  
Vol 5 (1) ◽  
pp. 545-548
Author(s):  
Stefan Scheuermann ◽  
Armin Schäfer ◽  
Jens Langejürgen ◽  
Christian Reis
Keyword(s):  
Single Cell ◽  
Stromal Cells ◽  
Processing Time ◽  
Single Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Tissue Samples ◽  
Tissue Dissociation ◽  
Tissue Matrix ◽  
Generation Sequencing

AbstractThe future of personalized diagnostics commences on the single cell level. Even high-end technologies like Next Generation Sequencing can be improved if applied on pure single cell populations (e.g., tumor cells without contaminating stromal cells) or on a single cell level (DNA/RNA sequencing). The vast majority of these technologies need individual and preferably undistorted cells for the analytical process. Thus, decisive prerequisite for high-end analytics is to remove cells from their tissue matrix as gently as possible. This can be accomplished by an enzyme-free, fast and reproducible approach of generating pure and individual single cells from tissue samples. In this study we demonstrate the utility of a semi-automated Tissue Grinder that is compatible with standard 50 ml centrifuge tubes and standard cell strainer for mechanically, nonenzymatic and parallel processing of tissue samples. We show that without enzymatic treatment viable single-cell yields match or exceed reference enzymatic methods, while reducing processing time by at least 80%.

scholarly journals Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells

Lab on a Chip ◽  
2018 ◽  
Vol 18 (18) ◽  
pp. 2776-2786 ◽  
Author(s):  
Xiaolong Qiu ◽  
Jeremy A. Lombardo ◽  
Trisha M. Westerhof ◽  
Marissa Pennell ◽  
Anita Ng ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Cell Suspensions ◽  
Cell Aggregates ◽  
Single Cell Level ◽  
Cell Level ◽  
Tissue Samples ◽  
Nylon Mesh

Tissues are increasingly being analyzed at this single cell level. We present a simple and inexpensive microfluidic filter device that can rapidly and effectively improve the quality of single cell suspensions obtained from digested tissue samples.

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 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 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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