Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells
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Abstract We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obatined by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.Keywords:
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Abstract We provide an overview of the methods used to label circulating cells for fluorescence detection by in vivo flow cytometry. These methods are useful for cell tracking in small animals without the need to draw blood samples and are particularly useful for the detection of circulating cancer cells and quantification of circulating immune cells. © 2011 International Society for Advancement of Cytometry.
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This chapter contains sections titled: The Cell and Cytometry Flow and Image Cytometry Chemical Cytometry Chemical Cytometry of DNA and mRNA Metabolic Cytometry Future Perspectives on Instrumentation for Chemical Cytometry Acknowledgments References
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Abstract Mass cytometry offers the advantage of allowing the simultaneous measurement of a greater number parameters than conventional flow cytometry. However, to date, mass cytometry has lacked a reliable alternative to the light scatter properties that are commonly used as a cell size metric in flow cytometry (forward scatter intensity—FSC). Here, we report the development of two plasma membrane staining assays to evaluate mammalian cell size in mass cytometry experiments. One is based on wheat germ agglutinin (WGA) staining and the other on Osmium tetroxide (OsO 4 ) staining, both of which have preferential affinity for cell membranes. We first perform imaging and flow cytometry experiments to establish a relationship between WGA staining intensity and traditional measures of cell size. We then incorporate WGA staining in mass cytometry analysis of human whole blood and show that WGA staining intensity has reproducible patterns within and across immune cell subsets that have distinct cell sizes. Lastly, we stain PBMCs or dissociated lung tissue with both WGA and OsO 4 ; mass cytometry analysis demonstrates that the two staining intensities correlate well with one another. We conclude that both WGA and OsO 4 may be used to acquire cell size‐related parameters in mass cytometry experiments, and expect these stains to be broadly useful in expanding the range of parameters that can be measured in mass cytometry experiments. © 2016 International Society for Advancement of Cytometry
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Abstract Simultaneous monitoring of biomarkers as well as single‐cell analyses based on flow cytometry and mass cytometry are important for investigations of disease mechanisms, drug discovery, and signaling‐network studies. Flow cytometry and mass cytometry are complementary to each other; however, probes that can satisfy all the requirements for these two advanced technologies are limited. In this study, we report a probe of lanthanide‐coordinated semiconducting polymer dots (Pdots), which possess fluorescence and mass signals. We demonstrated the usage of this dual‐functionality probe for both flow cytometry and mass cytometry in a mimetic cell mixture and human peripheral blood mononuclear cells as model systems. The probes not only offer high fluorescence signal for use in flow cytometry, but also show better performance in mass cytometry than the commercially available counterparts.
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Abstract Imaging flow cytometry shows significant potential for increasing our understanding of heterogeneous and complex life systems and is useful for biomedical applications. Ghost cytometry is a recently proposed approach for directly analyzing compressively measured signals of cells, thereby relieving a computational bottleneck for real‐time data analysis in high‐throughput imaging cytometry. In our previous work, we demonstrated that this image‐free approach could distinguish cells from two cell lines prepared with the same fluorescence staining method. However, the demonstration using different cell lines could not exclude the possibility that classification was based on non‐morphological factors such as the speed of cells in flow, which could be encoded in the compressed signals. In this study, we show that GC can classify cells from the same cell line but with different fluorescence distributions in space, supporting the strength of our image‐free approach for accurate morphological cell analysis. © 2020 International Society for Advancement of Cytometry
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Abstract Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are powerful tools for characterizing diverse tissues and cellular systems. Here mass cytometry was directly compared with fluorescence cytometry by studying phenotypes of healthy human peripheral blood mononuclear cells (PBMC) in the context of superantigen stimulation. One mass cytometry panel and five fluorescence cytometry panels were used to measure 20 well‐established lymphocyte markers of memory and activation. Comparable frequencies of both common and rare cell subpopulations were observed with fluorescence and mass cytometry using biaxial gating. The unsupervised high‐dimensional analysis tool viSNE was then used to analyze data sets generated from both mass and fluorescence cytometry. viSNE analysis effectively characterized PBMC using eight features per cell and identified similar frequencies of activated CD4+ T cells with both technologies. These results suggest combinations of unsupervised analysis programs and extended multiparameter cytometry will be indispensable tools for detecting perturbations in protein expression in both health and disease. © 2015 International Society for Advancement of Cytometry
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DNA measurements of 130 melanomas were carried out by flow cytometry (FCM) and image cytometry (ICM). ICM was applied to cytological preparations of fresh material (cICM) and to sections of formalin-fixed paraffin embedded tissue (sICM). The DNA ploidy, the DNA index of G0/G1 peaks (DI), and the proliferation index (PI) were used to compare all the methods. The following parameters reflecting malignancy were calculated only from ICM histograms: the 5c exceeding rate (5cER) and the malignancy grade (MG). In cases found to be DNA aneuploid by FCM, the PI values (FCM versus cICM) and the DIs (between all methods) showed a high correlation, and the concordance in relation to the DNA ploidy status was 96% (FCM versus cICM) and 94% (FCM versus sICM). However, we ascertained essential differences between FCM and ICM in melanomas classified as DNA diploid by FCM. The concordance in DNA ploidy was only 66% (FCM versus cICM) and 64% (FCM versus sICM). In contrast, cICM and sICM yielded similar results in most cases. With the exception of the near diploid range, ICM is superior to FCM in detecting DNA aneuploidy. In particular, DNA tetraploid stem lines can easily be overlooked by FCM. Therefore, DNA measurements of tumours judged to be DNA diploid by FCM must be verified by ICM. ICM on sections proved to be applicable and yielded reliable results provided that a suitable thickness was used, and the measuring of sectioned and overlapping nuclei was largely avoided by careful focusing in either direction. © 1996 Wiley-Liss, Inc.
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Simultaneous monitoring of biomarkers as well as single-cell analyses based on flow cytometry and mass cytometry are important for investigations of disease mechanisms, drug discovery, and signaling-network studies. Flow cytometry and mass cytometry are complementary to each other; however, probes that can satisfy all the requirements for these two advanced technologies are limited. In this study, we report a probe of lanthanide-coordinated semiconducting polymer dots (Pdots), which possess fluorescence and mass signals. We demonstrated the usage of this dual-functionality probe for both flow cytometry and mass cytometry in a mimetic cell mixture and human peripheral blood mononuclear cells as model systems. The probes not only offer high fluorescence signal for use in flow cytometry, but also show better performance in mass cytometry than the commercially available counterparts.
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Abstract We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obatined by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.
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