scholarly journals Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

10.3791/52201 ◽  
2014 ◽  
Author(s):  
Brian K. McFarlin ◽  
Adam S. Venable ◽  
Eric A. Prado ◽  
Andrea L. Henning ◽  
Randall R. Williams
Keyword(s):  
Flow Cytometry ◽  
Granulocyte Function

A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

1999 ◽  
Vol 19 (03) ◽  
pp. 134-138
Author(s):  
Gitta Kühnel ◽  
A. C. Matzdorff
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Blood Sample ◽  
Platelet Activation ◽  
Whole Blood ◽  
Receptor Occupancy ◽  
Aggregate Formation ◽  
Inhibitor Activity ◽  
Receptor Inhibitor

SummaryWe studied the effect of GPIIb/IIIa-inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GPIIb/IIIa-inhibitors (c7E3, DMP728, XJ757), then thrombin or ADP were added and after 1 min the sample was fixed. Samples without c7E3 but with 0.1 U/ml thrombin had a decrease in platelet count. Samples with increasing concentrations of c7E3 had a lesser or no decrease in platelet count. The two other inhibitors (DMP 725, XJ757) gave similar results. GPIIb/IIIa-inhibitors prevent aggregate formation and more single platelets remain in the blood sample. The agonist-induced decrease in platelet count correlates closely with the concentration of the GPIIb/IIIa inhibitor and receptor occupancy. This correlation may be used as a simple measure for inhibitor activity in whole blood.

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.

In Vitro Evaluation of Chitosan Induced Cytotoxicity against Cancerous Cell lines: MCF-7, Saos-2 and HeLa

2019 ◽  
Vol 19 ◽  
Author(s):  
Zeinab Abedian ◽  
Niloofar Jenabian ◽  
Ali Akbar Moghadamnia ◽  
Ebrahim Zabihi ◽  
Roghayeh Pourbagher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Anticancer Agents ◽  
Fibroblast Cells ◽  
Apoptosis Induction ◽  
Cytotoxic Effects ◽  
Cancerous Cell ◽  
Significant Concentration ◽  
Mcf 7

Objective/ Background: Cancer is still the most common cause of morbidity in world and new powerful anticancer agents without severe side effects from natural sources is important. Methods: The evaluation of cytotoxicity and apoptosis induction was carried out in MCF-7,HeLa and Saos-2 as cancerous cell lines with different histological origin and human fibroblast served as control normal cell. The cells were treated with different concentrations of chitosan and the cytotoxicity was determined using MTT assay after 24, 48 and 72 h .The mode of death was evaluated by flow cytometry . Results: While both types of chitosan showed significant concentration-dependently cytotoxic effects against the three cancerous cell lines, fibroblast cells showed somehow more compatibility with chitosan. On the other hand, there were no significant differences between LMWC and HMWC cytotoxicity in all cell lines. The flow cytometry results showed the apoptosis pattern of death more in Saos-2 and HeLa while necrosis was more observable with MCF7. Also higher viability with both types of chitosan was seen in fibroblast as normal cells Conclusion: Chitosan shows anticancerous effect against 3 cancerous cell lines, while it is compatible with normal diploid fibroblast cells. Furthermore, it seems that the molecular weight of chitosan does not affect its anticancerous property.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

Evaluating the Effect of Cinnarizine on Promastigotes and Amastigotes forms of Leishmania major

2020 ◽  
Vol 20 (4) ◽  
pp. 550-555 ◽  
Author(s):  
Lima Asgharpour Sarouey ◽  
Parvaneh Rahimi-Moghaddam ◽  
Fatemeh Tabatabaie ◽  
Khadijeh Khanaliha
Keyword(s):  
Flow Cytometry ◽  
Cutaneous Leishmaniasis ◽  
Leishmania Major ◽  
Inhibitory Effect ◽  
Control Group ◽  
Secondary Infections ◽  
Ic50 Values ◽  
J774 Cells ◽  
Mtt Assays

: As an important global disease, cutaneous leishmaniasis is associated with complications such as secondary infections and atrophic scars. The first line treatment with antimonials is expensive and reported to have serious side effects and enhance resistance development. The main objective of this study was to evaluate the effect of Cinnarizine on standard strains of Leishmania major because of paucity of information on this subject. Methods: In this experimental study, four concentrations of the drug (5, 10, 15 and 20 μg/ml) were added to Leishmania major cultures at 24, 48 and 72 hours intervals. MTT assays were performed to determine parasite viability and drug toxicity. Leishmania major promastigotes were augmented to the in vitro cultured macrophages (J774 cells) and then incubated for 72 hours. Half maximal inhibitory concentration (IC50) was ascertained by counting parasites. The inhibitory effect of the drug was compared with that of Glucantime. Flow-cytometry was performed to investigate apoptosis. Each test was repeated thrice. Results: The IC50 values of Cinnarizine after 72 hours were calculated to be 34.76 μg/ml and 23.73 μg/ml for promastigotes and amastigotes, respectively. The results of MTT assays showed 48 % promastigote viability after 72 hour-exposure to Cinnarizine at 20 μg/ml concentration. Programmed cell death in promastigote- and amastigote-infected macrophages was quantified to be 13.66 % and 98.7 %, respectively. Flow- cytometry analysis indicated that Cinnarizine induced early and late apoptosis in parasites. All treatments produced results which differed significantly from control group (P<0.05). Conclusion: Cinnarizine showed low toxicity with anti-leishmanial and apoptosis effects on both promastigote and intracellular amastigote forms. Therefore, we may suggest further assessment on animal models of this drug as candidates for cutaneous leishmaniasis therapy.

scholarly journals In Vitro Cellular Uptake Studies of Self-Assembled Fluorinated Nanoparticles Labelled with Antibodies

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1906
Author(s):  
Mona Atabakhshi-Kashi ◽  
Mónica Carril ◽  
Hossein Mahdavi ◽  
Wolfgang J. Parak ◽  
Carolina Carrillo-Carrion ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cellular Uptake ◽  
Hydrophobic Interactions ◽  
Intrinsic Properties ◽  
Wide Range ◽  
Self Assembled ◽  
Uptake Studies ◽  
Targeting Ability ◽  
Fluorinated Nanoparticles

Nanoparticles (NPs) functionalized with antibodies (Abs) on their surface are used in a wide range of bioapplications. Whereas the attachment of antibodies to single NPs to trigger the internalization in cells via receptor-mediated endocytosis has been widely studied, the conjugation of antibodies to larger NP assemblies has been much less explored. Taking into account that NP assemblies may be advantageous for some specific applications, the possibility of incorporating targeting ligands is quite important. Herein, we performed the effective conjugation of antibodies onto a fluorescent NP assembly, which consisted of fluorinated Quantum Dots (QD) self-assembled through fluorine–fluorine hydrophobic interactions. Cellular uptake studies by confocal microscopy and flow cytometry revealed that the NP assembly underwent the same uptake procedure as individual NPs; that is, the antibodies retained their targeting ability once attached to the nanoassembly, and the NP assembly preserved its intrinsic properties (i.e., fluorescence in the case of QD nanoassembly).

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Evaluation of the Expression of CCR5 and CX3CR1 Receptors and Correlation with the Functionality of T Cells in Women infected with ZIKV during Pregnancy

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Débora Familiar-Macedo ◽  
Iury Amancio Paiva ◽  
Jessica Badolato-Corrêa da Silva ◽  
Fabiana Rabe de Carvalho ◽  
Helver Gonçalves Dias ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Clinical Outcome ◽  
Cell Responses ◽  
Functional Assays ◽  
Asymptomatic Children ◽  
Cd107a Expression ◽  
Congenital Zika Syndrome ◽  
Ifn Γ

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child’s clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

scholarly journals Mechanistic Fingerprinting Reveals Kinetic Signatures of Resistance to Daptomycin and Host Defense Peptides in Streptococcus mitis-oralis

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 404
Author(s):  
Michael R. Yeaman ◽  
Liana C. Chan ◽  
Nagendra N. Mishra ◽  
Arnold S. Bayer
Keyword(s):  
Flow Cytometry ◽  
Host Defense ◽  
Durable Resistance ◽  
Cross Resistance ◽  
Streptococcus Mitis ◽  
Host Defense Peptides ◽  
Strain Pair ◽  
Defense Peptides

Streptococcus mitis-oralis (S. mitis-oralis) infections are increasingly prevalent in specific populations, including neutropenic cancer and endocarditis patients. S. mitis-oralis strains have a propensity to evolve rapid, high-level and durable resistance to daptomycin (DAP-R) in vitro and in vivo, although the mechanism(s) involved remain incompletely defined. We examined mechanisms of DAP-R versus cross-resistance to cationic host defense peptides (HDPs), using an isogenic S. mitis-oralis strain-pair: (i) DAP-susceptible (DAP-S) parental 351-WT (DAP MIC = 0.5 µg/mL), and its (ii) DAP-R variant 351-D10 (DAP MIC > 256 µg/mL). DAP binding was quantified by flow cytometry, in-parallel with temporal (1–4 h) killing by either DAP or comparative prototypic cationic HDPs (hNP-1; LL-37). Multicolor flow cytometry was used to determine kinetic cell responses associated with resistance or susceptibility to these molecules. While overall DAP binding was similar between strains, a significant subpopulation of 351-D10 cells hyper-accumulated DAP (>2–4-fold vs. 351-WT). Further, both DAP and hNP-1 induced cell membrane (CM) hyper-polarization in 351-WT, corresponding to significantly greater temporal DAP-killing (vs. 351-D10). No strain-specific differences in CM permeabilization, lipid turnover or regulated cell death were observed post-exposure to DAP, hNP-1 or LL-37. Thus, the adaptive energetics of the CM appear coupled to the outcomes of interactions of S. mitis-oralis with DAP and selected HDPs. In contrast, altered CM permeabilization, proposed as a major mechanism of action of both DAP and HDPs, did not differentiate DAP-S vs. DAP-R phenotypes in this S. mitis-oralis strain-pair.

scholarly journals MODL-12. DEVELOPMENT OF A NOVEL IMMUNOCOMPETENT MOUSE MODEL FOR DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii413-iii413
Author(s):  
Maggie Seblani ◽  
Markella Zannikou ◽  
Katarzyna Pituch ◽  
Liliana Ilut ◽  
Oren Becher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Growth Factor Receptor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Time Of Application ◽  
Tumor Associated Antigen ◽  
Sarcoma Virus

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor affecting young children. Immunotherapies hold promise however the lack of immunocompetent models recreating a faithful tumor microenvironment (TME) remains a challenge for development of targeted immunotherapeutics. We propose to generate an immunocompetent DIPG mouse model through induced overexpression of interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen overexpressed by glioma cells. A model with an intact TME permits comprehensive preclinical assessment of IL13Rα2-targeted immunotherapeutics. Our novel model uses the retroviral avian leucosis and sarcoma virus (RCAS) for in vivo gene delivery leading to IL13Rα2 expression in proliferating progenitor cells. Transfected cells expressing IL13Rα2 and PDGFB, a ligand for platelet derived growth factor receptor, alongside induced p53 loss via the Cre-Lox system are injected in the fourth ventricle in postnatal pups. We validated the expression of PDGFB and IL13Rα2 transgenes in vitro and in vivo and will characterize the TME through evaluation of the peripheral and tumor immunologic compartments using immunohistochemistry and flow cytometry. We confirmed expression of transgenes via flow cytometry and western blotting. Comparison of survival dynamics in mice inoculated with PDGFB alone with PDGFB+IL13Rα2 demonstrated that co-expression of IL13Rα2 did not significantly affect mice survival compared to the PDGFB model. At time of application, we initiated experiments to characterize the TME. Preliminary data demonstrate establishment of tumors within and adjacent to the brainstem and expression of target transgenes. Preclinical findings in a model recapitulating the TME may provide better insight into outcomes upon translation to clinical application.

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