scholarly journals Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

10.3791/58621 ◽  
2019 ◽  
Author(s):  
Jeffrey M. Finlon ◽  
Matthew A. Burchill ◽  
Beth A. Jirón Tamburini
Keyword(s):  
Endothelial Cells ◽  
Flow Cytometric Analysis ◽  
Lymphatic Endothelial Cells ◽  
Flow Cytometric ◽  
Murine Liver

Salidroside Inhibits Endogenous Hydrogen Peroxide Induced Cytotoxicity of Endothelial Cells

2013 ◽  
Vol 750-752 ◽  
pp. 1529-1532 ◽  
Author(s):  
Xing Yu Zhao ◽  
Lian Hai Jin ◽  
Dong Jun Wang ◽  
Bin Xu ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Glucose Oxidase ◽  
Flow Cytometric Analysis ◽  
Steady States ◽  
Protective Effects ◽  
Human Endothelial Cells ◽  
Flow Cytometric ◽  
Dose Dependent ◽  
Mtt Assays

To explore the protective effects of salidroside against endogenous hydrogen peroxide (H2O2) -induced cytotoxicity in human endothelial cells (EVC-304). EVC-304 cells were incubated in the presence or absence of low steady states of H2O2 (34μM) generated by glucose oxidase (GOX) with or without salidroside. MTT assays were performed, together with flow cytometric analysis using propidium (PI) label. The results indicated that salidroside could attenuate H2O2 induced cytotoxicity in EVC-304 cells in a dose-dependent pattern. Furthermore, flow cytometric analysis revealed that salidroside could also inhibited the G2/M arrest induced by endogenous hydrogen. The present study demonstrates that salidroside could inhibit endogenous hydrogen peroxide induced cytotoxicity of endothelial cells .

scholarly journals Interrogating the Interaction of CD52 Functionalised Metallic Nanoparticles with Malignant B Lymphocytes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4437-4437
Author(s):  
Laura Christine Kickham ◽  
Anthony M. McElligott ◽  
Adriele Prina-Mello ◽  
Elisabeth A. Vandenberghe ◽  
Yuri Volkov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Flow ◽  
B Lymphocytes ◽  
Flow Cytometric Analysis ◽  
Ph Sensitive ◽  
Fe2o3 Nanoparticles ◽  
Proof Of Concept ◽  
Fluid Shear ◽  
Flow Cytometric ◽  
Cytotoxicity Studies

Abstract Chronic Lymphocytic Leukaemia (CLL) is a common B-lymphoid malignancy with over 200,000 people affected annually in Europe and the US. The aim of therapy is to increase the quality and duration of life using well tolerated treatment. Novel intracellular drug delivery systems such as functionalised nanoparticles (NPs), conjugated to antibodies such as anti-CD20 or anti-CD52 directed at cell surface markers may help address this need. We have explored the feasibility of targeting nanoparticles in CLL using a microfluidics based adhesion assay, anti CD52 cell targeting and Fludarabine therapy. Methods B cells were isolated from the peripheral blood of normal healthy donors and CLL patients. The CLL cell line, I-83, was maintained under standard conditions. Epifluorescent, laser scanning confocal and electron microscopy was utilised for imaging the interaction of metallic NPs with cells. The metallic NPs were polymer-coated for biocompatibility and cellular toxicity was assessed using flow cytometric analysis based on changes in light scattering. NPs distribution on the surface of the cells was visualized using epifluorescent and Helium ion microscopy, cellular uptake and alterations in cell morphology after NP treatment was imaged by confocal microscopy. Cell adhesion and migration behaviour under fluid shear flow conditions mimicking CLL cells in vivo was investigated using a microfluidics system utilising biochips coated with VCAM-1 and seeded with Human Umbilical Vein Endothelial Cells (HUVEC) or Human Dermal Lymphatic Endothelial Cells. CD52-Alexa Fluor® 633 was conjugated to the surface of silanized NPs (NP1) using standard carbo-diimide cross linker chemistry techniques and successful functionalisation of NPs was validated using flow cytometric analysis, monitoring a shift in fluorescent population. I-83 cells and patient-derived malignant B cells were treated using pH sensitive dye doped NPs and pH sensitive dye doped NP1 in order to assess interaction of nanoparticles with cells. Uptake measurements were performed through quantification of the fluorescence of the pH sensitive dye. As proof of concept, Fludarabine was then incorporated on to the surface of NPs in order to investigate its potential as a nanotherapeutic. Cytotoxicity studies were performed using flow cytometric analysis mentioned above following a 24 hour incubation. Results and Conclusions Quantitative and qualitative analysis identified uptake of NPs by normal and malignant B-lymphocytes with optimal NPs concentration for uptake determined at 25 μg/ml. Non-functionalised NPs in the range of 15-50nm were internalised by cells. There was a notable decrease in the interaction of NPs with cells under physiologically relevant fluid shear flow in comparison to static conditions, resulting in a corresponding decrease in uptake, highlighting the rationale for a CLL cell-targeted NP. The results of the adhesion experiment using I-83 cells and patient derived CLL cells to the HUVEC monolayer in a micro-fluidics system showed that patient CLL adhesion decreased after NP treatment (p=0.01, n=3). Cytotoxicity studies show that exposure to uncoated Fe2O3 nanoparticles yields an IC50 value of 23μg/mL +/- 5 μg/mL in comparison to coated, stabilized Fe2O3 nanoparticles with an IC50 of 49μg/mL +/- 5 μg/mL. Functionalisation of NPs with CD52 antibody (NP-1) resulted in significantly increased uptake (p<0.0001, n=3) and cytotoxicity. Preparation of these nanoparticles was reproducible and the particles remained stable in suspension for over 4 weeks. Cells treated with NPs bound Fludarabine were found to have significantly increased cytotoxicity in comparison to stabilized NPs (IC50 of 21μg/mL +/- 1μg/mL. In summary, this work provides proof of concept of efficacy for a targeted nanotherapeutic in haematological malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Appearance of claudin-5+ leukocyte subtypes in the blood and CNS during progression of EAE

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Dylan Krajewski ◽  
Debayon Paul ◽  
Shujun Ge ◽  
Evan Jellison ◽  
Joel S. Pachter
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Demyelinating Disease ◽  
Flow Cytometric Analysis ◽  
Autoimmune Encephalomyelitis ◽  
Flow Cytometric ◽  
Leukocyte Transendothelial Migration ◽  
Transient Interactions ◽  
Circulating T Cells ◽  
Experimental Autoimmune

Abstract Background Tight junctions (TJs) are membrane specializations characteristic of barrier-forming membranes, which function to seal the aqueous pathway between endothelial cells or epithelial cells and, thereby, obstruct intercellular solute and cellular movement. However, previous work from our laboratory found that claudin-5 (CLN-5), a TJ protein prominent at the blood–brain barrier (BBB), was also detected, ectopically, on leukocytes (CLN-5+) in the blood and central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory, demyelinating disease that is a model for multiple sclerosis. CLN-5 was further shown to be transferred from endothelial cells to circulating leukocytes during disease, prompting consideration this action is coupled to leukocyte transendothelial migration (TEM) into the CNS by fostering transient interactions between corresponding leukocyte and endothelial junctional proteins at the BBB. Methods To begin clarifying the significance of CLN-5+ leukocytes, flow cytometry was used to determine their appearance in the blood and CNS during EAE. Results Flow cytometric analysis revealed CLN-5+ populations among CD4 and CD8 T cells, B cells, monocytes and neutrophils, and these appeared with varying kinetics and to different extents in both blood and CNS. CLN-5 levels on circulating T cells further correlated highly with activation state. And, the percentage of CLN-5+ cells among each of the subtypes analyzed was considerably higher in CNS tissue than in blood, consistent with the interpretation that CLN-5+ leukocytes gain preferred access to the CNS. Conclusion Several leukocyte subtypes variably acquire CLN-5 in blood before they enter the CNS, an event that may represent a novel mechanism to guide leukocytes to sites for paracellular diapedesis across the BBB.

Isolation and flow cytometric analysis of the free lymphomyeloid cells present in murine liver

1990 ◽  
Vol 132 (1) ◽  
pp. 137-144 ◽  
Author(s):  
Pierre L. Goossens ◽  
Hélène Jouin ◽  
Gilles Marchal ◽  
Geneviève Milon
Keyword(s):  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Murine Liver

scholarly journals Establishment of Blood Outgrowth Endothelial Cells from a Type 3 Von Willebrand Disease (VWD) Patient with a Neutralizing Anti-VWF Inhibitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1508-1508 ◽  
Author(s):  
Naomi Sanda ◽  
Nobuaki Suzuki ◽  
Mayuko Kishimoto ◽  
Natsumi Kameyama ◽  
Yoko Kajiura ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Dna Sequences ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Nonsense Mutations ◽  
Flow Cytometric ◽  
Type 3 ◽  
Von Willebrand

Abstract Background: Type 3 von Willebrand disease (VWD) is characterized by a complete loss type of von Willebrand Factor (VWF) with the rarest disease frequency and hemorrhagic symptom is the most severe among other VWD types. The development of alloantibodies directed against VWF occurs in approximately 10% of patients with type 3 VWD. In this study, we found the VWF gene alterations and established blood outgrowth endothelial cells (BOECs) a from a Japanese type 3 VWD patient with an anti-VWF inhibitors. Case: A 5-year-old woman suffered from epistaxis, purpura and easy bruising and has been diagnosed as VWD. Her plasma level of FVIII:C was 1.8% and VWF:Ag and VWF:RCo was <5% and <10%, respectively. Her plasma lacked VWF multimers, indicating that she had type 3 VWD. To treat her frequent muco-cutaneous bleeding, purified plasma-derived FVIII/VWF concentrates were administrated followed by inhibitor development. After about 20ED of FVIII/VWF concentrates, an anaphylactic symptom comprised of cough, dyspnea, and wheezing developed at the time of dosing. An high titer inhibitory antibody against VWF was confirmed by a Bethesda assay based on a assay of VWF:RCo. Her younger sister also had type 3 VWD. Methods: Patients samples were collected after the written informed consent has bee obtained. We performed MLPA for genetic deletion or insertion. Then we amplified the all exons including the exon/intron boundaries of the VWF gene by PCR using allele-specific primers, and analyzed DNA sequences of the patient. Peripheral blood mononuclear cells were obtained from the patient and BOECs were then established. Briefly, buffy coat mononuclear cells were isolated and seeded onto a 6-well tissue culture plate precoated with type 1 rat tail collagen at 37°C, 5% CO2, in a humidified incubator. Medium was changed daily for 7 days and then every other day until the first passage. The endothelial identity of the BOECs was confirmed by flow cytometric and immunofluorescence analyses using endothelial markers with antibodies to CD31, VE-Cadherin (CD144) and VEGFR-2 (CD309). Results: The large deletion and insertion of VWF gene were not found. Direct sequencing showed the propositus and her sister were compound heterozygous for an E2341X (c.7021G>T) mutation in exon41 and a Y2631X (c.7892-7893insA) mutation in exon48. These were therefore two novel nonsense mutations and normal VWF polypeptides could not be translated. Flow cytometric analysis indicated that established BOECs expressed cell surface CD31 and CD309, whereas CD34 was not detected. By immunofluorescence analysis of fixed BOECs, VWF signal was remarkably reduced from the patient, in spite of normal VE-Cadherin expression. VWF specific ELISA was performed for both cell supernatant and lysates of patient’s BOECs but immunodetectable VWF was not secreted. Discussion: We identified two novel nonsense mutations causing type3 VWD. BOECs established from the patient reproduced the phenotype of the disease, suggesting that BOEC analysis is useful for studying the molecular pathogenesis of von Willebrand disease. Disclosures No relevant conflicts of interest to declare.

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