| 1. |
- Adler, Jeremy, et al.
(författare)
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Quantifying Colocalization by Correlation : The Pearson Correlation Coefficient is Superior to the Mander's Overlap Coefficient
- 2010
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Ingår i: CYTOMETRY PART A. - : Wiley. - 1552-4922 .- 1552-4930. ; 77A:8, s. 733-742
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Tidskriftsartikel (refereegranskat)abstract
- The Pearson correlation coefficient (PCC) and the Mander's overlap coefficient (MOC) are used to quantify the degree of colocalization between fluorophores. The MOC was introduced to overcome perceived problems with the PCC. The two coefficients are mathematically similar, differing in the use of either the absolute intensities (MOC) or of the deviation from the mean (PCC). A range of correlated datasets, which extend to the limits of the PCC, only evoked a limited response from the MOC. The PCC is unaffected by changes to the offset while the MOC increases when the offset is positive. Both coefficients are independent of gain. The MOC is a confusing hybrid measurement, that combines correlation with a heavily weighted form of co-occurrence, favors high intensity combinations, downplays combinations in which either or both intensities are low and ignores blank pixels. The PCC only measures correlation. A surprising finding was that the addition of a second uncorrelated population can substantially increase the measured correlation, demonstrating the importance of excluding background pixels. Overall, since the MOC is unresponsive to substantial changes in the data and is hard to interpret, it is neither an alternative to nor a useful substitute for the PCC. The MOC is not suitable for making measurements of colocalization either by correlation or co-occurrence.
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| 2. |
- Adler, Jeremy, et al.
(författare)
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Quantifying Colocalization: the Case for Discarding the Manders Overlap Coefficient.
- 2021
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Ingår i: Cytometry. Part A : the journal of the International Society for Analytical Cytology. - : Wiley. - 1552-4930. ; 99:9, s. 910-920
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Tidskriftsartikel (refereegranskat)abstract
- Colocalization measurements aim to characterize the relative distribution of two molecules within a biologically relevant area. It is efficient to measure two distinct features, co-occurrence, the extent to which the molecules appear together, and correlation, how well variations in concentration of the two molecules match. The Manders overlap coefficient (MOC) appears in most colocalization software but the literature contains three interpretations of its measurements: a) co-occurrence, b) correlation or c) a combination of both. This is surprising given the simplicity of the underlying equation. Testing shows that the MOC responds both to changes in co-occurrence and to changes in correlation. Further testing reveals that different distributions of intensity (Gaussian, gamma, uniform, exponential) dramatically alter the balance between the contribution from co-occurrence and correlation. It follows that the MOC's ability to differentiate between different patterns of colocalization is very limited, since any value is compatible with widely differing combinations of co-occurrence, correlation and intensity distribution. To characterize colocalization we recommend reporting both co-occurrence and correlation, using coefficients specific for each attribute. Since the MOC has no clear role in the measurement of colocalization and causes considerable confusion, we conclude that it should be discarded. This article is protected by copyright. All rights reserved.
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| 3. |
- Allalou, Amin, et al.
(författare)
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Robust signal detection in 3D fluorescence microscopy
- 2010
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Ingår i: Cytometry. Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 77A:1, s. 86-96
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Tidskriftsartikel (refereegranskat)abstract
- Robust detection and localization of biomolecules inside cells is of great importance to better understand the functions related to them. Fluorescence microscopy and specific staining methods make biomolecules appear as point-like signals on image data, often acquired in 3D. Visual detection of such point-like signals can be time consuming and problematic if the 3D images are large, containing many, sometimes overlapping, signals. This sets a demand for robust automated methods for accurate detection of signals in 3D fluorescence microscopy. We propose a new 3D point-source signal detection method that is based on Fourier series. The method consists of two parts, a detector, which is a cosine filter to enhance the point-like signals, and a verifier, which is a sine filter to validate the result from the detector. Compared to conventional methods, our method shows better robustness to noise and good ability to resolve signals that are spatially close. Tests on image data show that the method has equivalent accuracy in signal detection in comparison to Visual detection by experts. The proposed method can be used as an efficient point-like signal detection tool for various types of biological 3D image data.
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| 4. |
- Antoniadi, Ioanna, et al.
(författare)
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Best practices in plant cytometry
- 2021
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 99, s. 311-317
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Tidskriftsartikel (refereegranskat)
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| 5. |
- Antoniadi, Ioanna, et al.
(författare)
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Fluorescence activated cell sorting-A selective tool for plant cell isolation and analysis
- 2022
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 101, s. 725-736
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Tidskriftsartikel (refereegranskat)abstract
- Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.
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| 6. |
- Apweiler, Rolf, et al.
(författare)
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Approaching clinical proteomics : current state and future fields of application in cellular proteomics
- 2009
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Ingår i: Cytometry. Part A : the journal of the International Society for Analytical Cytology. - : Wiley. - 1552-4922. ; 75A:10, s. 816-832
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Forskningsöversikt (refereegranskat)abstract
- Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.
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| 7. |
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| 8. |
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| 9. |
- Bengtsson, Ewert, et al.
(författare)
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Special Section on Image Cytometry
- 2019
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 95A:4, s. 363-365
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 10. |
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| 11. |
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| 12. |
- Cieslar-Pobuda, Artur, et al.
(författare)
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Cell Type Related Differences in Staining with Pentameric Thiophene Derivatives
- 2014
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 85A:7, s. 628-635
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent compounds capable of staining cells selectively without affecting their viability are gaining importance in biology and medicine. Recently, a new family of optical dyes, denoted luminescent conjugated oligothiophenes (LCOs), has emerged as an interesting class of highly emissive molecules for studying various biological phenomena. Properly functionalized LCOs have been utilized for selective identification of disease-associated protein aggregates and for selective detection of distinct cells. Herein, we present data on differential staining of various cell types, including cancer cells. The differential staining observed with newly developed pentameric LCOs is attributed to distinct side chain functionalities along the thiophene backbone. Employing flow cytometry and fluorescence microscopy we examined a library of LCOs for stainability of a variety of cell lines. Among tested dyes we found promising candidates that showed strong or moderate capability to stain cells to different extent, depending on target cells. Hence, LCOs with diverse imidazole motifs along the thiophene backbone were identified as an interesting class of agents for staining of cancer cells, whereas LCOs with other amino acid side chains along the backbone showed a complete lack of staining for the cells included in the study. Furthermore, for p-HTMI,a LCO functionalized with methylated imidazole moieties, the staining was dependent on the p53 status of the cells, indicating that the molecular target for the dye is a cellular component regulated by p53. We foresee that functionalized LCOs will serve as a new class of optical ligands for fluorescent classification of cells and expand the toolbox of reagents for fluorescent live imaging of different cells.
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Feridani, Amir, et al.
(författare)
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Combined flow cytometry and confocal laser scanning microscopy for evaluation of BR96 antibody cancer cell targeting and internalization.
- 2007
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 71A:6, s. 361-370
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Tidskriftsartikel (refereegranskat)abstract
- Background: Monoclonal antibodies (mAb) are important tools in the management of tumor disease, and the discovery of antibodies with both specific cancer cell targeting and capacity to enter the cells by internalization are critical to improve the therapeutic efficacy. Method: Antibody cancer cell targeting and internalization properties of fluoroscein-conjugated mAb made against Lewis Y (BR96) were evaluated quantitatively and qualitatively by means of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM), respectively, on cells from a rat tumor cell line (BN7005-H1D2). Results: The study demonstrated a specific binding of BR96 to LewisY (LeY) located in the cell membrane and as BR96/LeY immunocomplexes (BR96/LeY) internalized into the cytoplasm. BR96/LeY was internalized into about 15% of the cells, usually distributed throughout the cytoplasm, but also located close to the nuclei. Cytotoxic effects by BR96 were indicated, and CLSM visualized subpopulations containing cells with bound or internalized BR96/LeY that possessed morphologically pyknotic nuclei and disrupted DNA. Conclusion: The spatial-temporal pattern by BR96 cell targeting and internalization processes of BR96/LeY into the cancer cells expressing LeY was demonstrated by FCM and CLSM. Used together, the FCM and CLSM techniques provide a valuable tool for preclinical analyses of antibody targeting and their capacities as carriers of cytotoxic conjugates for the use in cancer therapy.
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| 17. |
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| 18. |
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| 19. |
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| 20. |
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| 21. |
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| 22. |
- Gréen,, Anna, 1973-, et al.
(författare)
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Translocation of Histone H1 Subtypes Between Chromatin and Cytoplasm During Mitosis in Normal Human Fibroblasts
- 2010
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 77A:5, s. 478-484
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Tidskriftsartikel (refereegranskat)abstract
- Histone H1 is an important constituent of chromatin which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3 and H1.5 during mitosis. H1.2 was found in chromatin during prophase, and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase it appeared in both chromatin and cytoplasm, and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but some H1.5 remained situated in chromatin during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or their phosphorylated variants, may be signalling molecules in mitosis or that they leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.
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| 23. |
- Grenvall, Carl, et al.
(författare)
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Label-free somatic cell cytometry in raw milk using acoustophoresis.
- 2012
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 81A:12, s. 1076-1083
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Tidskriftsartikel (refereegranskat)abstract
- A microfluidic system for cell enumeration in raw milk was developed. The new method, preconditions the milk sample using acoustophoresis that removes lipid particles which are larger than a few micrometers. The acoustophoretic preprocessing eliminates the need for conventional sample preparation techniques, which include chemical solvents, cell labeling and centrifugation, and facilitates rapid cell enumeration using microscopy or coulter counter measurements. By introducing an acoustic standing wave with three pressure nodes in a microchannel at the same time as the milk sample is laminated to the channel center, lipids are acoustically driven to the closest pressure antinode at each side of the channel center and the cells in the milk sample are focused in the central pressure node. The extracted center fraction with cells becomes sufficiently clean from lipid vesicles to enable enumeration of somatic cells without any labeling step either by direct light microscopy or by coulter counting. Obtained lipid free milk fractions clearly revealed the cell fraction when analyzed by Coulter Counting. Cell counting as measured by a Coulter Counter after acoustophoretic lipid depletion aligned with the corresponding data obtained by reference measurements based on fluorescence staining and subsequent flow cytometer analysis. © 2012 International Society for Advancement of Cytometry.
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| 24. |
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| 25. |
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| 26. |
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| 27. |
- Koch, Christin, et al.
(författare)
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CHIC - An automated approach for the detection of dynamic variations in complex microbial communities
- 2013
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 83A:6, s. 561-567
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Tidskriftsartikel (refereegranskat)abstract
- Altering environmental conditions change structures of microbial communities. These effects have an impact on the single-cell level and can be sensitively detected using community flow cytometry. However, although highly accurate, microbial monitoring campaigns are still rarely performed applying this technique. One reason is the limited access to pattern analysis approaches for the evaluation of microbial cytometric data. In this article, a new analyzing tool, Cytometric Histogram Image Comparison (CHIC), is presented, which realizes trend interpretation of variations in microbial community structures (i) without any previous definition of gates, by working (ii) person independent, and (iii) with low computational demand. Various factors influencing a sensitive determination of changes in community structures were tested. The sensitivity of this technique was found to discriminate down to 0.5% internal variation. The final protocol was exemplarily applied to a complex microbial community dataset, and correlations to experimental variation were successfully shown.
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| 28. |
- Kostova-Koleva, Nora N., et al.
(författare)
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Histone H5chromatin interactions in situ are strongly modulated by H5 C-terminal phosphorylation
- 2013
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Ingår i: Cytometry Part A. - : Wiley-Blackwell. - 1552-4922 .- 1552-4930. ; 83A:3, s. 273-279
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Tidskriftsartikel (refereegranskat)abstract
- We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones.
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| 29. |
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| 30. |
- Larsson, Sara, et al.
(författare)
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A Markov model approach shows a large variation in the length of S phase in MCF-7 breast cancer cells
- 2005
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 65A:1, s. 15-25
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Tidskriftsartikel (refereegranskat)abstract
- Background: The potential doubling time of a tumor has been suggested to be a measurement of tumor aggressiveness; therefore, it is of interest to find reliable methods to estimate this time. Because of variability in length of the various cell cycle phases, stochastic modeling of the cell cycle might be a suitable approach. Methods: The relative movement curve and the DNA synthesis time were estimated by using local polynomial regression methods. Further, the rate of nucleotide incorporation was estimated by using a Markov pure birth process with one absorbing state to model the progression of the DNA distribution through S phase. Results: An estimate of the DNA synthesis time, with confidence intervals, was obtained from the relative movement curve. The Markov approach provided an estimate of the distribution of the time to complete S phase given the initial distribution. Using the Markov approach we also made an estimate of the mean number of active replicons during S phase. Conclusions: A Markov pure birth process has shown to be useful to model the progression of cells through S phase and to increase knowledge about the variability in the length of S phase and a large variation is shown.
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| 31. |
- Lenshof, Andreas, et al.
(författare)
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Efficient Purification of CD4+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Affinity Bead Acoustophoresis
- 2014
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 85A:11, s. 933-941
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Tidskriftsartikel (refereegranskat)abstract
- Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity-bead-mediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell-bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n = 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti-CD4-coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ lymphocytes demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead-labeled CD4+ lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinity-bead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology
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| 32. |
- Leuchowius, Karl-Johan, et al.
(författare)
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Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family
- 2009
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Ingår i: Cytometry: Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 75A:10, s. 833-839
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Tidskriftsartikel (refereegranskat)abstract
- Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo- and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amounts of interactions between EGFR and HER2, but the amount of dimerization was not found to be affected significantly upon stimulation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immunofluorescence staining. In situ PLA was successfully used to study receptor dimerization and activation of the EGF-receptor family with high selectivity and sensitivity. The combination of in situ PLA and flow cytometry provided a statistically powerful way of analyzing protein-protein interactions and post-translational modifications on a single-cell basis.
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| 33. |
- Magnusson, Karin, et al.
(författare)
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Differential vital staining of normal fibroblasts and melanoma cells by an anionic conjugated polyelectrolyte
- 2015
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Ingår i: Cytometry Part A. - : Wiley: 12 months. - 1552-4922 .- 1552-4930. ; 87:3, s. 262-272
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Tidskriftsartikel (refereegranskat)abstract
- Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes.
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| 34. |
- Malm, Patrik, et al.
(författare)
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Simulation of bright-field microscopy images depicting pap-smear specimen
- 2015
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 87:3, s. 212-226
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Tidskriftsartikel (refereegranskat)abstract
- As digital imaging is becoming a fundamental part of medical and biomedical research, the demand for computer-based evaluation using advanced image analysis is becoming an integral part of many research projects. A common problem when developing new image analysis algorithms is the need of large datasets with ground truth on which the algorithms can be tested and optimized. Generating such datasets is often tedious and introduces subjectivity and interindividual and intraindividual variations. An alternative to manually created ground-truth data is to generate synthetic images where the ground truth is known. The challenge then is to make the images sufficiently similar to the real ones to be useful in algorithm development. One of the first and most widely studied medical image analysis tasks is to automate screening for cervical cancer through Pap-smear analysis. As part of an effort to develop a new generation cervical cancer screening system, we have developed a framework for the creation of realistic synthetic bright-field microscopy images that can be used for algorithm development and benchmarking. The resulting framework has been assessed through a visual evaluation by experts with extensive experience of Pap-smear images. The results show that images produced using our described methods are realistic enough to be mistaken for real microscopy images. The developed simulation framework is very flexible and can be modified to mimic many other types of bright-field microscopy images.
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| 35. |
- Nilsson, Björn, et al.
(författare)
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Segmentation of complex cell clusters in microscopic images: Application to bone marrow samples. : application to bone marrow samples
- 2005
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 66A:1, s. 24-31
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Morphologic examination of bone marrow and peripheral blood samples continues to be the cornerstone in diagnostic hematology. In recent years, interest in automatic leukocyte classification using image analysis has increased rapidly. Such systems collect a series of images in which each cell must be segmented accurately to be classified correctly. Although segmentation algorithms have been developed for sparse cells in peripheral blood, the problem of segmenting the complex cell clusters characterizing bone marrow images is harder and has not been addressed previously.METHODS: We present a novel algorithm for segmenting clusters of any number of densely packed cells. The algorithm first oversegments the image into cell subparts. These parts are then assembled into complete cells by solving a combinatorial optimization problem in an efficient way.RESULTS: Our experimental results show that the algorithm succeeds in correctly segmenting densely clustered leukocytes in bone marrow images.CONCLUSIONS: The presented algorithm enables image analysis-based analysis of bone marrow samples for the first time and may also be adopted for other digital cytometric applications where separation of complex cell clusters is required.
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| 36. |
- Olm, Franziska, et al.
(författare)
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Acoustophoresis enables the label-free separation of functionally different subsets of cultured bone marrow stromal cells
- 2021
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Ingår i: Cytometry. Part A : the journal of the International Society for Analytical Cytology. - : Wiley. - 1552-4930. ; 99:5, s. 476-487
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Tidskriftsartikel (refereegranskat)abstract
- Culture-expanded mesenchymal stromal cells (MSCs) are promising candidates for clinical cell-based therapies. MSC products are heterogeneous and we therefore investigated whether acoustophoresis, an ultrasound-based separation technology, could be used for the label-free enrichment of functionally different MSC populations. Acoustophoresis uses an ultrasonic standing wave in a microchannel which differentially affects the movement of cells depending on their acoustophysical properties, such as size, density, and compressibility. Human bone marrow MSCs were generated by standard adherent culture in xenofree medium and separated by microchip acoustophoresis. MSCs with up to 20% higher proliferation and 1.7-fold increased clonogenic potential were enriched in the side outlet of the chip compared to the input sample. These cells were significantly smaller (average diameter 14.5 ± 0.4 μm) compared to the center outlet fraction (average diameter 17.1 ± 0.6 μm) and expressed higher levels of genes related to proliferation and stem cell properties (i.e. Ki-67 (1.9-fold), Nanog1 (6.65-fold), Oct4 (2.9-fold), and CXCL12 (1.8-fold), n = 3) in the side outlet compared to input. Fractions of MSCs in G0 /G1 cell cycle phase were significantly enriched in the side fraction and an up to 2.8-fold increase of cells in S/G2 /M phases were observed in center fractions compared to side fractions and 1.3-fold increased compared to the input sample. Acoustophoresis did not compromise MSC phenotype, proliferation, clonogenic capacity, and viability (generally 87-98%), nor did it affect differentiation or immunomodulatory capacities. These results demonstrate that label-free acoustic separation can enrich functionally different MSC subsets which can potentially be employed to produce better-defined stromal cell products from cultured MSCs. Hence, acoustophoresis is a potentially promising separation technology to provide improved cell products for research and possible future clinical use.
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| 37. |
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| 38. |
- Pinidiyaarachchi, Amalka, et al.
(författare)
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A detailed analysis of 3D subcellular signal localization
- 2009
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 75A:4, s. 319-328
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Tidskriftsartikel (refereegranskat)abstract
- Detection and localization of fluorescent signals in relation to other subcellular structures is an important task in various biological studies. Many methods for analysis of fluorescence microscopy image data are limited to 2D. As cells are in fact 3D structures, there is a growing need for robust methods for analysis of 3D data. This article presents an approach for detecting point-like fluorescent signals and analyzing their subnuclear position. Cell nuclei are delineated using marker-controlled (seeded) 3D watershed segmentation. User-defined object and background seeds are given as input, and gradient information defines merging and splitting criteria. Point-like signals are detected using a modified stable wave detector and localized in relation to the nuclear membrane using distance shells. The method was applied to a set of biological data studying the localization of Smad2-Smad4 protein complexes in relation to the nuclear membrane. Smad complexes appear as early as 1 min after stimulation while the highest signal concentration is observed 45 min after stimulation, followed by a concentration decrease. The robust 3D signal detection and concentration measures obtained using the proposed method agree with previous observations while also revealing new information regarding the complex formation.
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| 39. |
- Pontén, Olle, et al.
(författare)
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PACMan : A software package for automated single-cell chlorophyll fluorometry
- 2024
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 105:3, s. 203-213
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Tidskriftsartikel (refereegranskat)abstract
- Microalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand-operated pulse-amplitude-modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations- or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY-PAM system, we present the PAM Automation Control Manager (‘PACMan’), an open-source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components. PACMan comes with a user-friendly graphical user interface and is released together with a stand-alone tool (PAMalysis) for the automated calculation of per-cell maximum quantum efficiencies (= Fv/Fm). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genus Symbiodiniaceae) within custom-made microfluidic devices. Compared to the manual operation of MICROSCOPY-PAM systems, this represents a 10-fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY-PAM system to repeatedly capture high-quality image data across multiple positions. Finally, we analyzed single-cell Fv/Fm with the manufacturer-supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand-operated MICROSCOPY-PAM technologies.
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| 40. |
- Ranefall, Petter, 1968-, et al.
(författare)
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Global Gray-level Thresholding Based on Object Size
- 2016
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 89A:4, s. 385-390
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Tidskriftsartikel (refereegranskat)abstract
- In this article, we propose a fast and robust global gray-level thresholding method based on object size, where the selection of threshold level is based on recall and maximum precision with regard to objects within a given size interval. The method relies on the component tree representation, which can be computed in quasi-linear time. Feature-based segmentation is especially suitable for biomedical microscopy applications where objects often vary in number, but have limited variation in size. We show that for real images of cell nuclei and synthetic data sets mimicking fluorescent spots the proposed method is more robust than all standard global thresholding methods available for microscopy applications in ImageJ and CellProfiler. The proposed method, provided as ImageJ and CellProfiler plugins, is simple to use and the only required input is an interval of the expected object sizes.
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| 41. |
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| 42. |
- Rockenbauer, Eszter, et al.
(författare)
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SNP genotyping using microsphere-linked PNA and flow cytometric detection
- 2005
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 64A:2, s. 80-86
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Tidskriftsartikel (refereegranskat)abstract
- Background: Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene. Methods: Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allele-specific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and immobilized PNA was carried out at 60 degrees C followed by flow cytometric detection. Results: We present a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection used for the correct and fast regenotyping of a Danish basal cell carcinoma cohort. Conclusions: This new assay presents a simple, rapid, and robust method for SNP genotyping for laboratories equipped with a standard flow cytometer. Moreover, this system offers potential for multiplexing and will be operational for middle-scale genotyping.
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| 43. |
- Rundberg Nilsson, Alexandra, et al.
(författare)
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Frequency determination of rare populations by flow cytometry: A hematopoietic stem cell perspective.
- 2013
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 83A:8, s. 721-727
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Forskningsöversikt (refereegranskat)abstract
- Flow cytometry allows for identification of cellular subsets based on cell intrinsic properties, most often by the use of fluorochrome-conjugated antibodies recognizing distinct cell-surface epitopes that define the cells of interest. Advances in technical instrumentation and the availability of an ever-increasing number of fluorophores, today enables identification of multicolor defined cellular populations to a previously unreachable resolution. However, these possibilities put an increasing demand on preparation, acquisition, and subsequent analysis of the investigated samples. Identification of very rare cellular subsets, such as the bone marrow-residing hematopoietic stem cells (HSCs), causes further complexity to such analysis. Here, we discuss considerations and aspects in multicolor flow cytometry as exemplified by analysis of human and mouse HSCs. We illustrate advantages and drawbacks of polychromatic flow cytometry and propose strategies, such as the use of internal reference populations, for sample analysis. © 2013 International Society for Advancement of Cytometry.
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| 44. |
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| 45. |
- Rönnlund, Daniel, et al.
(författare)
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Spatial organization of proteins in metastasizing cells
- 2013
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 83:9, s. 855-865
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Tidskriftsartikel (refereegranskat)abstract
- The ability of tumor cells to invade into the surrounding tissue is linked to defective adhesive and mechanical properties of the cells, which are regulated by cell surface adhesions and the intracellular filamentous cytoskeleton, respectively. With the aim to further reveal the underlying mechanisms and provide new strategies for early cancer diagnostics, we have used ultrahigh resolution stimulated emission depletion (STED) microscopy as a means to identify metastasizing cells, based on their subcellular protein distribution patterns reflecting their specific adhesive and mechanical properties. We have compared the spatial distribution of cell-matrix adhesion sites and the vimentin filamentous systems in a matched pair of primary, normal, and metastatic human fibroblast cells. We found that the metastatic cells showed significantly increased densities and more homogenous distributions of nanoscale adhesion-related particles. Moreover, they showed an increase in the number but reduced sizes of the areas of cell-matrix adhesion complexes. The organization of the vimentin intermediate filaments was also found to be significantly different in the metastasizing cells, showing an increased entanglement and loss of directionality. Image analysis procedures were established, allowing an objective detection and characterization of these features and distinction of metastatic cells from their normal counterparts. In conclusion, our results suggest that STED microscopy provides a novel tool to identify metastasizing cells from a very sparse number of cells, based on the altered spatial distribution of the cell-matrix adhesions and intermediate filaments.
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| 46. |
- Sandstedt, Mikael, 1990, et al.
(författare)
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Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.
- 2015
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Ingår i: Cytometry. Part A : the journal of the International Society for Analytical Cytology. - : Wiley. - 1552-4930. ; 87:12, s. 1079-1089
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Tidskriftsartikel (refereegranskat)abstract
- Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples. © 2015 International Society for Advancement of Cytometry.
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| 47. |
- Snipstad, Sofie, et al.
(författare)
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Labeling nanoparticles : Dye leakage and altered cellular uptake
- 2017
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 91:8, s. 760-766
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Tidskriftsartikel (refereegranskat)abstract
- In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake, and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues. To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose, we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4 and 37C. Cell-NP interaction is confirmed by cellular fluorescence after 37C incubation, and NP-dye retention is confirmed when no cellular fluorescence is detected at 4C. Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing NP-based applications. © 2016 International Society for Advancement of Cytometry.
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| 48. |
- Solorzano, Leslie, 1989-, et al.
(författare)
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Machine learning for cell classification and neighborhood analysis in glioma tissue
- 2021
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 99:12, s. 1176-1186
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Tidskriftsartikel (refereegranskat)abstract
- Multiplexed and spatially resolved single-cell analyses that intend to study tissue heterogeneity and cell organization invariably face as a first step the challenge of cell classification. Accuracy and reproducibility are important for the downstream process of counting cells, quantifying cell-cell interactions, and extracting information on disease-specific localized cell niches. Novel staining techniques make it possible to visualize and quantify large numbers of cell-specific molecular markers in parallel. However, due to variations in sample handling and artifacts from staining and scanning, cells of the same type may present different marker profiles both within and across samples. We address multiplexed immunofluorescence data from tissue microarrays of low-grade gliomas and present a methodology using two different machine learning architectures and features insensitive to illumination to perform cell classification. The fully automated cell classification provides a measure of confidence for the decision and requires a comparably small annotated data set for training, which can be created using freely available tools. Using the proposed method, we reached an accuracy of 83.1% on cell classification without the need for standardization of samples. Using our confidence measure, cells with low-confidence classifications could be excluded, pushing the classification accuracy to 94.5%. Next, we used the cell classification results to search for cell niches with an unsupervised learning approach based on graph neural networks. We show that the approach can re-detect specialized tissue niches in previously published data, and that our proposed cell classification leads to niche definitions that may be relevant for sub-groups of glioma, if applied to larger data sets.
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| 49. |
- Talebizadeh, Nooshin, 1977-, et al.
(författare)
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Objective automated quantification of fluorescence signal in histological sections of rat lens
- 2017
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 91:8, s. 815-821
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Tidskriftsartikel (refereegranskat)abstract
- Visual quantification and classification of fluorescent signals is the gold standard in microscopy. The purpose of this study was to develop an automated method to delineate cells and to quantify expression of fluorescent signal of biomarkers in each nucleus and cytoplasm of lens epithelial cells in a histological section. A region of interest representing the lens epithelium was manually demarcated in each input image. Thereafter, individual cell nuclei within the region of interest were automatically delineated based on watershed segmentation and thresholding with an algorithm developed in Matlab™. Fluorescence signal was quantified within nuclei, cytoplasms and juxtaposed backgrounds. The classification of cells as labelled or not labelled was based on comparison of the fluorescence signal within cells with local background. The classification rule was thereafter optimized as compared with visual classification of a limited dataset. The performance of the automated classification was evaluated by asking 11 independent blinded observers to classify all cells (n = 395) in one lens image. Time consumed by the automatic algorithm and visual classification of cells was recorded. On an average, 77% of the cells were correctly classified as compared with the majority vote of the visual observers. The average agreement among visual observers was 83%. However, variation among visual observers was high, and agreement between two visual observers was as low as 71% in the worst case. Automated classification was on average 10 times faster than visual scoring. The presented method enables objective and fast detection of lens epithelial cells and quantification of expression of fluorescent signal with an accuracy comparable with the variability among visual observers.
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| 50. |
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