scholarly journals Imaging flow cytometry documents incomplete resistance of human sickle F-cells to ex vivo hypoxia-induced sickling

Blood ◽  
2014 ◽  
Vol 124 (4) ◽  
pp. 658-660 ◽  
Author(s):  
Kleber Yotsumoto Fertrin ◽  
Eduard J. van Beers ◽  
Leigh Samsel ◽  
Laurel G. Mendelsohn ◽  
Rehan Saiyed ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ex Vivo ◽  
Imaging Flow Cytometry ◽  
F Cells ◽  
Incomplete Resistance

scholarly journals Characterization of neutrophils and macrophages from ex vivo-cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry

Methods ◽  
2017 ◽  
Vol 112 ◽  
pp. 124-146 ◽  
Author(s):  
Margery G.H. Pelletier ◽  
Klaudia Szymczak ◽  
Anna M. Barbeau ◽  
Gianna N. Prata ◽  
Kevin S. O’Fallon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Ex Vivo ◽  
Functional Responses ◽  
Murine Bone Marrow ◽  
Imaging Flow Cytometry

Imaging Flow Cytometry Documents Incomplete Resistance Of F-Cells To Hypoxia-Induced Sickling In Blood Samples From Patients With Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 183-183
Author(s):  
Kleber Yotsumoto Fertrin ◽  
Eduard J. van Beers ◽  
Leigh Samsel ◽  
Laurel G. Mendelsohn ◽  
Rehan Saiyed ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Red Blood Cells ◽  
Cell Count ◽  
Sickle Cell Anemia ◽  
Blood Cells ◽  
Blood Samples ◽  
Imaging Flow Cytometry ◽  
Hypoxic Conditions ◽  
Round Shape ◽  
F Cells

Abstract Fetal hemoglobin (HbF) production induced by hydroxyurea is the mainstay of treatment for sickle cell anemia (SCA). Increased HbF production correlates with a higher number of HbF-containing red blood cells (RBCs) called F-cells. Successful treatment with hydroxyurea is associated with an increased number of F-cells, less hemolysis, improvement of anemia, and decreased frequency of vaso-occlusive crises in SCA patients. Comparison of in vitro sickling among blood specimens from sickle mouse models and from patients with different HbF levels has provided compelling evidence that increasing the percentage of circulating F-cells is associated with improvement of hemolytic biomarkers. While it has been demonstrated that higher HbF content prolongs sickle RBC survival, there is only indirect evidence of the response to hypoxia of F-cells compared to non-F-cells. We investigated the influence of HbF content on sickling through our recently developed Sickle Imaging Flow Cytometry Assay (SIFCA). SIFCA allows simultaneous analysis of both expression of intracellular proteins and morphological features of each cell in a robust, reproducible, operator-independent sickling assay. Peripheral venous blood samples were collected upon written consent from adult SCA patients with a wide range of HbF percentages (HbF range 2.0-26.9%) (n=15, nine on hydroxyurea treatment). RBC pellets were used to prepare 1% suspensions that were subjected to deoxygenation for 2 hours at 2% oxygen. RBCs were then labeled for HbF using a standard protocol for F-cell quantitation and a minimum of 20,000 cells were analyzed by imaging flow cytometry (ImageStreamX Mk II, Amnis Corporation), allowing the correlation between shape change and intensity of HbF expression for each RBC. We confirmed previous observations using conventional flow cytometry that F-cell count percentages significantly correlate with mean HbF determined by HPLC (r2P=0.9700, 95% CI 0.9098-0.9902, P<0.0001). F-cell count by SIFCA correlated highly with conventional F-cell flow cytometry by an independent CLIA-certified facility (r2P =0.9976, 95% CI 0.9861-0.9996, P<0.0001). SIFCA morphological analysis showed that the percentage of non-F-cells sickling upon deoxygenation was significantly higher than among F-cells (17.75% [95% CI 12.5-23.00] vs. 12.41% [95% CI 8.67-16.15], P=0.0015), a 1.498-fold difference (95% CI 1.228-1.768). Image analysis also allowed us to identify the presence of F-cells that still sickle despite their high HbF content, as well as non-F-cells that are resistant to sickling (Figure 1). Transmission electron microscopy of F-cells enriched by fluorescence activated cell sorting confirmed that sickled F-cells contained hemoglobin S polymers. In summary, we have documented for the first time at the individual RBC level that human F-cells are less prone to sickle under hypoxia ex vivo than non-F-cells. This study also illustrates the power of imaging flow cytometry to characterize predisposition to sickling in populations of red blood cells from the same patient, and would be suitable for use as a supportive biomarker assay in clinical trials investigating the efficacy of novel HbF inducers and their anti-sickling effect in a single assay. While the finding that F-cells sickle less than non-F-cells is not unexpected, it seems surprising to us that the difference in hypoxia-induced sickling between F-cells and non-F-cells is so small. This finding emphasizes the need to characterize additional RBC features that render individual cells more susceptible or resistant to sickling. Identification of factors besides HbF that modulate sickle hemoglobin polymerization may help design novel therapies for hydroxyurea-resistant SCA patients.Figure 1Sample images showing non-F-cells (left column) and F-cells (right column) as they appear on imaging flow cytometry. Under hypoxic conditions, non-F-cells are expected to sickle (panel A), while F-cells are expected to maintain a round shape (panel B). Nevertheless, round erythrocytes can be found among non-F-cells (panel C), as well as typically sickled F-cells (panel D).Figure 1. Sample images showing non-F-cells (left column) and F-cells (right column) as they appear on imaging flow cytometry. Under hypoxic conditions, non-F-cells are expected to sickle (panel A), while F-cells are expected to maintain a round shape (panel B). Nevertheless, round erythrocytes can be found among non-F-cells (panel C), as well as typically sickled F-cells (panel D). Disclosures: No relevant conflicts of interest to declare.

Synthesis of a Novel Curcumin Derivative as a Potential Imaging Probe in Alzheimer’s Disease Imaging

2019 ◽  
Vol 16 (8) ◽  
pp. 723-731 ◽  
Author(s):  
Alexander Sturzu ◽  
Sumbla Sheikh ◽  
Hubert Kalbacher ◽  
Thomas Nägele ◽  
Christopher Weidenmaier ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Flow Cytometry ◽  
Transgenic Mice ◽  
Ex Vivo ◽  
Amyloid Plaques ◽  
Laser Scanning Microscopy ◽  
Β Amyloid ◽  
Curcumin Derivative

Background: Curcumin has been of interest in the field of Alzheimer’s disease. Early studies on transgenic mice showed promising results in the reduction of amyloid plaques.However, curcumin is very poorly soluble in aqueous solutions and not easily accessible to coupling as it contains only phenolic groups as potential coupling sites. For these reasons only few imaging studies using curcumin bound as an ester were performed and curcumin is mainly used as nutritional supplement. Methods: In the present study we produced an aminoethyl ether derivative of curcumin using a nucleophilic substitution reaction. This is a small modification and should not impact the properties of curcumin while introducing an easily accessible reactive amino group. This novel compound could be used to couple curcumin to other molecules using the standard methods of peptide synthesis. We studied the aminoethyl-curcumin compound and a tripeptide carrying this aminoethyl-curcumin and the fluorescent dye fluorescein (FITC-curcumin) in vitro on cell culture using confocal laser scanning microscopy and flow cytometry. Then these two substances were tested ex vivo on brain sections prepared from transgenic mice depicting Alzheimer-like β-amyloid plaques. Results: In the in vitro CLSM microscopy and flow cytometry experiments we found dot-like unspecific uptake and only slight cytotoxicity correlating with this uptake. As these measurements were optimized for the use of fluorescein as dye we found that the curcumin at 488nm fluorescence excitation was not strong enough to use it as a fluorescence marker in these applications. In the ex vivo sections CLSM experiments both the aminoethyl-curcumin and the FITC-curcumin peptide bound specifically to β- amyloid plaques. Conclusion: In conclusion we successfully produced a novel curcumin derivative which could easily be coupled to other imaging or therapeutic molecules as a sensor for amyloid plaques.

scholarly journals Semi-automated classification of colonial Microcystis by FlowCAM imaging flow cytometry in mesocosm experiment reveals high heterogeneity during seasonal bloom

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yersultan Mirasbekov ◽  
Adina Zhumakhanova ◽  
Almira Zhantuyakova ◽  
Kuanysh Sarkytbayev ◽  
Dmitry V. Malashenkov ◽  
...  
Keyword(s):  
Machine Learning ◽  
Flow Cytometry ◽  
Spatial Resolution ◽  
Mesocosm Experiment ◽  
Imaging Flow Cytometry ◽  
Leibler Divergence ◽  
Temporal And Spatial ◽  
High Level ◽  
Training Sets

AbstractA machine learning approach was employed to detect and quantify Microcystis colonial morphospecies using FlowCAM-based imaging flow cytometry. The system was trained and tested using samples from a long-term mesocosm experiment (LMWE, Central Jutland, Denmark). The statistical validation of the classification approaches was performed using Hellinger distances, Bray–Curtis dissimilarity, and Kullback–Leibler divergence. The semi-automatic classification based on well-balanced training sets from Microcystis seasonal bloom provided a high level of intergeneric accuracy (96–100%) but relatively low intrageneric accuracy (67–78%). Our results provide a proof-of-concept of how machine learning approaches can be applied to analyze the colonial microalgae. This approach allowed to evaluate Microcystis seasonal bloom in individual mesocosms with high level of temporal and spatial resolution. The observation that some Microcystis morphotypes completely disappeared and re-appeared along the mesocosm experiment timeline supports the hypothesis of the main transition pathways of colonial Microcystis morphoforms. We demonstrated that significant changes in the training sets with colonial images required for accurate classification of Microcystis spp. from time points differed by only two weeks due to Microcystis high phenotypic heterogeneity during the bloom. We conclude that automatic methods not only allow a performance level of human taxonomist, and thus be a valuable time-saving tool in the routine-like identification of colonial phytoplankton taxa, but also can be applied to increase temporal and spatial resolution of the study.

scholarly journals High-throughput multiparametric imaging flow cytometry: toward diffraction-limited sub-cellular detection and monitoring of sub-cellular processes

Cell Reports ◽  
2021 ◽  
Vol 34 (10) ◽  
pp. 108824
Author(s):  
Gregor Holzner ◽  
Bogdan Mateescu ◽  
Daniel van Leeuwen ◽  
Gea Cereghetti ◽  
Reinhard Dechant ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Multiparametric Imaging ◽  
Cellular Processes ◽  
Imaging Flow Cytometry

scholarly journals Conventional, High-Resolution and Imaging Flow Cytometry: Benchmarking Performance in Characterisation of Extracellular Vesicles

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 124
Author(s):  
Jaco Botha ◽  
Haley R. Pugsley ◽  
Aase Handberg
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Extracellular Vesicles ◽  
Single Particles ◽  
Multiple Parameters ◽  
Imaging Flow Cytometry ◽  
Highly Sensitive ◽  
Individual Project ◽  
Benchmarking Performance ◽  
High Throughput Manner

Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.

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