Cytometry. Part A : the journal of the International Society for Analytical Cytology
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Những tiến bộ gần đây trong kỹ thuật nhuộm nội bào, công nghệ đo tế bào, thuốc nhuộm huỳnh quang, và sản xuất kháng thể đã mở rộng số lượng kháng nguyên nội bào có thể phân tích bằng phương pháp đo dòng tế bào. Việc đo lường phosphoryl hoá protein với kháng thể đặc hiệu phospho đã mang lại cái nhìn sâu sắc về các chuỗi tín hiệu kinase. Tuy nhiên, những kỹ thuật hiện có về nhuộm phospho-epitope có thể khác nhau nhiều, khiến việc hiểu rõ sự khác biệt giữa các kết quả ứng dụng các kỹ thuật này và phát triển các phương pháp ứng dụng bền vững, có thể tái lập là cần thiết.
Mười kỹ thuật cố định tế bào và làm cho tế bào thẩm thấu khác nhau đã được kiểm tra về khả năng cung cấp nhuộm đặc hiệu phospho. Các tổ hợp formaldehyde, methanol, ethanol, acetone, Triton X-100, và saponin được sử dụng như các chất cố định và thẩm thấu. Các kháng thể đặc hiệu phospho được gắn thuốc nhuộm Alexa Fluor để cung cấp phân tích đa sắc màu của các sự kiện tín hiệu khác nhau đồng thời trong từng tế bào.
Cố định tế bào với 1,5% formaldehyde sau đó làm thẩm thấu trong methanol đã cho kết quả tối ưu cho nhuộm pERK, pp38, pJNK, pStat1, pStat5, và pStat6. Thay đổi thời gian cố định formaldehyde và thẩm thấu methanol làm ảnh hưởng đến đo lường sự kích hoạt phosphoryl hoá. Phân tích đo dòng tế bào đặc hiệu phospho có sự tương quan tốt với phương pháp Western blotting, cung cấp sự xác thực nền tảng chéo cho kỹ thuật này.
Việc đo sự kiện phosphoryl hoá bằng phương pháp đo dòng tế bào cung cấp một cách nhanh chóng và hiệu quả để đo chuỗi kinase trong từng tế bào. Sự ổn định của phospho-epitope trong methanol cho phép lưu trữ lâu dài các mẫu trước khi phân tích. Nhiều chuỗi tín hiệu có thể được giám sát đồng thời thông qua việc sử dụng các nhãn thuốc nhuộm huỳnh quang khác nhau để xác định sự đặc hiệu của các ligand hoặc chất ức chế. Áp dụng các kỹ thuật được tối ưu hóa cho các loại tế bào không đồng nhất như máu ngoại vi hoặc tế bào lách chuột có thể cho phép phân tích tín hiệu đồng thời trong các tập hợp tế bào miễn dịch. Cytometry Part A 55A:61–70, 2003. © 2003 Wiley-Liss, Inc.
The wound healing assay is a commonly used technique to measure cell motility and migration. Traditional methods of performing the wound healing assay suffer from low throughput and a lack of quantitative data analysis. We have developed a new method to perform a high‐throughput wound healing assay that produces quantitative data using the LEAP™ instrument. The LEAP™ instrument is used to create reproducible wounds in each well of a 96‐well plate by laser ablation. The LEAP™ then records bright field images of each well at several time points. A custom texture segmentation algorithm is used to determine the wound area of each well at each time point. This texture segmentation analysis can provide faster and more accurate image analysis than traditional methods. Experimental results show that reproducible wounds are created by laser ablation with a wound area that varies by less than 10%. This method was tested by confirming that neuregulin‐2β increases the rate of wound healing by MCF7 cells in a dose dependent manner. This automated wound healing assay has greatly improved the speed and accuracy, making it a suitable high‐throughput method for drug screening. © 2011 International Society for Advancement of Cytometry
Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C‐reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7β‐hydroxycholesterol, 7‐ketocholesterol, and 25‐hydroxycholesterol), and various cytokines.
Different flow cytometric bead‐based assays were used to quantify some cytokines (IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐8, IL‐10, IL‐12, IL‐13, IL‐17, G‐CSF, GM‐CSF, IFN‐γ, MCP‐1, MIP‐1β, or TNF‐α) in the culture media of oxysterol‐treated U937 and THP‐1 cells, and in the sera of healthy and atherosclerotic subjects. CRP and markers of hyperlipidemia were determined with routine analytical methods. Oxysterols were quantified by gas chromatography/mass spectrometry. Flow cytometric and biochemical methods were used to measure IL‐8 mRNA levels, intracellular IL‐8 content, and protein phosphorylation in the mitogenic extracellular kinase/extracellular signal‐regulated kinase1/2 (MEK/ERK1/2) signaling pathway.
All oxysterols investigated are potent in vitro inducers of MCP‐1, MIP‐1β, TNF‐α, and/or IL‐8 secretion, the latter involving the MEK/ERK1/2 cell signaling pathway. In healthy and atherosclerotic subjects, no relationships were found between cytokines (IL‐8, IL‐1β, IL‐6, IL‐10, TNF‐α, IL‐12, and MCP‐1), CRP, conventional markers of hyperlipidemia, and oxysterols. However, in patients with arterial disorders of the lower limbs, small but statistically significant differences in the circulating levels of CRP, TNF‐α, and IL‐10 were observed comparatively to healthy subjects and according to the atherosclerotic stage considered.
Flow cytometric bead‐based assays are well adapted to measure variations of cytokine secretion in the culture media of oxysterol‐treated cells and in the sera of healthy and atherosclerotic subjects. They underline the in vitro proinflammatory properties of oxysterols and may permit to distinguish healthy and atherosclerotic subjects, as well as various atherosclerotic stages. © 2006 International Society for Analytical Cytology
The aim of this article is to perform a statistical analysis of reactive oxygen species (ROS) cytometric data. It is demonstrated that the classical parametric and nonparametric statistical tests are not suitable to examine these data; the Kolmogorov‐Smirnov test and the modification proposed by Lampariello are shown to be too sensitive with respect to the experimental bias (due to procedure or to the instrument) and variability in the ROS production within the repeated samples. Several approaches are examined and discussed. Modifications of the Lampariello's procedure are proposed to include the variability within samples. The validity of the proposed approach is verified by analyzing repeated measurements of ROS formation in cultured human lymphocytes untreated or treated with ferrous sulfate. The proposed approach is successful in considering the “intersample” variability in the ROS data analysis and keeps a good level of validity. Nevertheless, this procedure is not user‐friendly and needs to be handled by an expert operator. © 2007 International Society for Analytical Cytology
Immunophenotyping of blood lymphocyte subsets and activation markers is a basic tool in the diagnostic process of primary immunodeficiency diseases, its use becoming more and more widespread as the knowledge about these illnesses increases. However, the availability of reliable reference values, which need to be age‐matched for the pediatric population, is a pre‐requisite for the reliable interpretation of immunophenotyping data. Aim of this study is to analyze the lymphocyte subsets and activation markers distribution in children aged 0–18 years referring to the University Hospital of Padova and to create age‐matched reference values expressed by percentiles, thus providing a valuable guideline for the interpretation of the immunophenotype. © 2014 International Society for Advancement of Cytometry
Spectral imaging extends the capabilities of biological and clinical studies to simultaneously study multiple features such as organelles and proteins qualitatively and quantitatively. Spectral imaging combines two well‐known scientific methodologies, namely spectroscopy and imaging, to provide a new advantageous tool. The need to measure the spectrum at each point of the image requires combining dispersive optics with the more common imaging equipment, and introduces constrains as well.
The principles of spectral imaging and a few representative applications are described. Spectral imaging analysis is necessary because the complex data structure cannot be analyzed visually. A few of the algorithms are discussed with emphasis on the usage for different experimental modes (fluorescence and bright field). Finally, spectral imaging, like any method, should be evaluated in light of its advantages to specific applications, a selection of which is described.
Spectral imaging is a relatively new technique and its full potential is yet to be exploited. Nevertheless, several applications have already shown its potential. © 2006 International Society for Analytical Cytology
The study of T cell biology has been accelerated by substantial progress at the technological level, particularly through the continuing advancement of flow cytometry. The conventional approach of observing T cells as either T helper or T cytotoxic is overly simplistic and does not allow investigators to clearly identify immune mechanisms or alterations in physiological processes that impact on clinical outcomes. The complexity of T cell sub‐populations, as we understand them today, combined with the immunological and functional diversity of these subsets represent significant complications for the study of T cell biology. In this article, we review the use of classical markers in delineating T cell sub‐populations, from “truly naïve” T cells (recent thymic emigrants with no proliferative history) to “exhausted senescent” T cells (poorly proliferative cells that display severe functional abnormalities) wherein the different phenotypes of these populations reflect their disparate functionalities. In addition, since persistent infections and chronological aging have been shown to be associated with significant alterations in human T cell distribution and function, we also discuss age‐associated and cytomegalovirus‐driven alterations in the expression of key subset markers. © 2013 International Society for Advancement of Cytometry
In recent years, a tremendous effort has been devoted to the detailed characterization of the phenotype and function of distinct T cell subpopulations in humans, as well as to their pathway(s) of differentiation and role in immune responses. But these studies seem to have generated more questions than definitive answers. To clarify issues related to the function and differentiation of T cell subsets, one session of the MASIR 2008 conference was dedicated to this topic. Several points of consensus and discord were highlighted in the work presented during this session. We provide here an account of these points, including the relative heterogeneity of T cell subpopulations during infections with distinct pathogens, the relationship between phenotypic and functional T cell attributes, and the pathway(s) of T cell differentiation. Finally, we discuss the problems which still limit general agreement. Published 2008 Wiley‐Liss, Inc.
The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen.
The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI.
Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 ± 0.004. The uncertainty in the final RI value represents the technique measurement error.
The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology. © 2005 Wiley‐Liss, Inc.
Evaluation of the potential hazard of man‐made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO2 nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human‐derived retinal pigment epithelial cells (ARPE‐19) were incubated with TiO2 nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 μg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur™ flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO2. From 0.1 to 30 μg/ml TiO2, the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO2 particles. Based on the parameters of morphology and the calcein‐AM/propidium iodide viability assay, TiO2 concentrations below 30 μg/ml TiO2 caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E‐800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO2 (0.1–0.3 μg/ml), the flow cytometer could detect as few as 5–10 nanoparticles per cell due to intense light scattering by TiO2. Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health. Published 2010 Wiley‐Liss, Inc.
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