scholarly journals Fast and Simple Tool for the Quantification of Biofilm-Embedded Cells Sub-Populations from Fluorescent Microscopic Images

2017 ◽  
Author(s):  
Mikhail I Bogachev ◽  
Vladimir Yu Volkov ◽  
Oleg A Markelov ◽  
Elena Yu Trizna ◽  
Diana R Baydamshina ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Software Tool ◽  
Laser Scanning Microscopy ◽  
Cell Counting ◽  
Eukaryotic Cells ◽  
Distribution Analysis ◽  
Cell Size Distribution ◽  
Suggested Approach ◽  
Microscopic Images

AbstractFluorescent staining is a common tool for both quantitative and qualitative assessment of pro- and eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell counting by flow cytometry is often limited, for example when working with cells rigidly adhered either to each other or to external surfaces like bacterial biofilms or adherent cell lines and tissue samples. An alternative approach is provided by using fluorescent microscopy and confocal laser scanning microscopy (CLSM), which enables the evaluation of fractions of cells subpopulations in a given sample. For the quantitative assessment of cell fractions in microphotographs, we suggest a simple two-step algorithm that combines single cells selection and the statistical analysis. To facilitate the first step, we suggest a simple procedure that supports finding the balance between the detection threshold and the typical size of single cells based on objective cell size distribution analysis. Based on a series of experimental measurements performed on bacterial and eukaryotic cells under various conditions, we show explicitly that the suggested approach effectively accounts for the fractions of different cell sub-populations (like the live/dead staining in our samples) in all studied cases that are in good agreement with manual cell counting on microphotographs and flow cytometry data. This algorithm is implemented as a simple software tool that includes an intuitive and user-friendly graphical interface for the initial adjustment of algorithm parameters to the microphotographs analysis as well as for the sequential analysis of homogeneous series of similar microscopic images without further user intervention. The software tool entitled BioFilmAnalyzer is freely available online at https://bitbucket.org/rogex/biofilmanalyzer/downloads/.

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 Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kazufumi Sakamoto ◽  
Yoshitsune Hondo ◽  
Naoki Takahashi ◽  
Yuhei Tanaka ◽  
Rikuto Sekine ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Kuramoto Model ◽  
Distribution Analysis ◽  
Cell Clusters ◽  
Overdrive Suppression ◽  
The Kuramoto Model

AbstractWe investigated the dominant rule determining synchronization of beating intervals of cardiomyocytes after the clustering of mouse primary and human embryonic-stem-cell (hES)-derived cardiomyocytes. Cardiomyocyte clusters were formed in concave agarose cultivation chambers and their beating intervals were compared with those of dispersed isolated single cells. Distribution analysis revealed that the clusters’ synchronized interbeat intervals (IBIs) were longer than the majority of those of isolated single cells, which is against the conventional faster firing regulation or “overdrive suppression.” IBI distribution of the isolated individual cardiomyocytes acquired from the beating clusters also confirmed that the clusters’ IBI was longer than those of the majority of constituent cardiomyocytes. In the complementary experiment in which cell clusters were connected together and then separated again, two cardiomyocyte clusters having different IBIs were attached and synchronized to the longer IBIs than those of the two clusters’ original IBIs, and recovered to shorter IBIs after their separation. This is not only against overdrive suppression but also mathematical synchronization models, such as the Kuramoto model, in which synchronized beating becomes intermediate between the two clusters’ IBIs. These results suggest that emergent slower synchronous beating occurred in homogeneous cardiomyocyte clusters as a community effect of spontaneously beating cells.

Synergistic Effects of 5-Fluorouracil in Combination with Diosmetin in Colorectal Cancer Cells

2021 ◽  
Vol 7 (1) ◽  
pp. 6
Author(s):  
Sareh Kamran ◽  
Ajantha Sinniah ◽  
Mohammed Abdullah Alshawsh
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Synergistic Effect ◽  
Cancer Cells ◽  
Synergistic Effects ◽  
Neoadjuvant Treatment ◽  
Software Tool ◽  
New Combination ◽  
Combination Regimen ◽  
Colorectal Cancer Cells

Colorectal cancer (CRC) is among the most commonly occurring cancers. The management of CRC includes laparoscopic surgery, radiotherapy, chemotherapies and neoadjuvant treatment. However, conventional chemotherapies have poor impact on combating CRC and are associated with severe toxic effects and high rates of relapse. Therefore, searching for a new combination regimen is a favorable consideration. The aim of this study was to elucidate the synergistic effect of 5-fluorouracil (5-FU) and diosmetin in an in vitro model on colorectal cancer cells. An MTT assay was conducted on HCT-116 cancer cells and they were treated with a concentration gradient of 5-FU and diosmetin individually and in combination. The combination index (CI) and dose reduction index (DRI) were calculated using CompuSyn software. Isobologram analysis and synergism determination were performed using the Combenefit software tool and the synergy score was calculated using the SynergyFinder 2.0 software tool. The apoptotic features of the cells were determined via an AO/PI double staining assay and an annexin V assay using a fluorescent microscope and the flow cytometry technique, respectively. The findings showed that the DRI of 5-FU was three-fold lower in the combination with a CI value of less than one, which indicates that there was a synergistic effect. The AO/PI microscopic results revealed signs of apoptosis and dead cells after 72 h of treatment. Flow cytometry analysis confirmed that the apoptotic effect of the combination was more prominent compared to 5-FU alone. The findings of this study offer a potential strategy for reducing the cytotoxicity and enhancing the efficacy of 5-FU on colorectal cancer cells through a synergistic study model.

Analysis of ploidy level of Artemisia annua L. based on flow cytometry and confocal laser scanning microscopy

2021 ◽  
Vol 50 (1) ◽  
pp. 29-35
Author(s):  
Shu-Gen Wei ◽  
Ling-Yun Wan ◽  
Ying Wei ◽  
Li-Li He ◽  
Jin-E Fu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Artemisia Annua ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Ploidy Levels ◽  
Artemisia Annua L

Eighty nine Artemisia samples treated with different concentrations of colchicine were used as breeding samples, with diploid Artemisia as the control. The ploidy levels of samples were determined by flow cytometry and confocal laser scanning microscopy (CLSM). An analysis of the flow cytometry results identified three suspected tetraploid plants and seven suspected triploid plants. The results of this study may be useful for breeding new Artemisia lines.

Fast intracellular pH determination in single cells by flow-cytometry

1981 ◽  
Vol 68 (5) ◽  
pp. 265-266 ◽  
Author(s):  
G. Valet ◽  
A. Raffael ◽  
L. Moroder ◽  
E. W�nsch ◽  
G. Ruhenstroth-Bauer
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph ◽  
Single Cells

scholarly journals Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0240769
Author(s):  
Prasanna Channathodiyil ◽  
Jonathan Houseley
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cyclin B1 ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Simple Method ◽  
Staining Methods

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

scholarly journals Therapeutic Effect of Exogenous Treg Cells on Collagen-Induced Arthritis and Rheumatoid Arthritis

2019 ◽  
Author(s):  
Shutong Li ◽  
Hongxing Wang ◽  
Hui Wu ◽  
Guoqing Zhang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Synovial Fibroblast ◽  
Treg Cells ◽  
Cell Counting ◽  
Fibroblast Cells ◽  
Collagen Induced Arthritis ◽  
Gm Csf

Abstract Background Regulatory T (Treg) cells have anti-inflammatory and anti-autoimmune functions. The proportion and functions of Treg cells are perturbed in rheumatoid arthritis (RA) patients. Methods Human Treg cells were induced to amplify in vitro and cocultured with RA synovial fibroblast cells (RASFs). The proliferation and apoptosis of RASFs were determined by the cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Human Treg cells were also injected to collagen-induced arthritis (CIA) rats via the tail vein. Changes in lymphocyte subtypes and cytokines in the peripheral blood and spleen were observed by flow cytometry. Results After coculture with the Treg cells, the proliferation of RA synovial fibroblast cells decreased (p<0.01), and the rate of apoptosis increased (p=0.037). The human Treg cells were injected into the tail veins of collagen-induced arthritis (CIA) rats. The severity of the CIA was reduced (p<0.01) following the injection, the percentages of rat endogenous Treg cells in the peripheral blood and spleen increased significantly (p=0.007 and p<0.01, respectively), and the proportion of B cells decreased (p=0.031). The levels of interleukin IL-5 and IL-6 and the Th1/Th2 ratio in the peripheral blood were significantly decreased (p=0.013, 0.009 and 0.012, respectively). The number of NK cells and the levels of IL-4, IL-13, TNF-α, IFN-γ and GM-CSF in the peripheral blood and spleen did not change significantly. Conclusion These results suggest that exogenous Treg cells play a therapeutic role in RA and CIA. Treg cell treatment could serve as a therapy for RA.

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