The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique

Cytometry ◽  
2001 ◽  
Vol 47 (1) ◽  
pp. 24-31 ◽  
Author(s):  
L.F.R. Span ◽  
A.H.M. Pennings ◽  
G. Vierwinden ◽  
J.B.M. Boezeman ◽  
R.A.P. Raymakers ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dynamic Process ◽  
Annexin V

scholarly journals Evaluation of Immobilization of Selected Peat-Isolated Yeast Strains of the Species Candida albicans and Candida subhashii on the Surface of Artificial Support Materials Used for Biotrickling Filtration

Processes ◽  
2020 ◽  
Vol 8 (7) ◽  
pp. 801
Author(s):  
Milena Marycz ◽  
Anna Brillowska-Dąbrowska ◽  
Jacek Gębicki
Keyword(s):  
Flow Cytometry ◽  
Candida Albicans ◽  
Propidium Iodide ◽  
Annexin V ◽  
Support Materials ◽  
Hydrophobic Compounds ◽  
Yeast Strains ◽  
Different Types ◽  
Materials Used ◽  
Hydrophilic And Hydrophobic Compounds

The paper describes the process of n-butanol abatement by unicellular fungi, able to deplete n-butanol content in gas, by using n-butanol as source of carbon. Isolated and identified fungi species Candida albicans and Candida subhashii were subjected to a viability process via assimilation of carbon from hydrophilic and hydrophobic compounds. The isolates, which exhibited the ability to assimilate carbon, were immobilized on four different types of artificial support materials used for biotrickling filtration. Application of optical microscopy, flow cytometry and the tests employing propidium iodide and annexin V revealed viability of the fungi isolated on support materials’ surfaces at the average level of 95%. The proposed method of immobilization and its evaluation appeared to be effective, cheap and fast. Based on performed comparative analyses, it was shown that polyurethane foam and Bialecki rings (25 × 25) could be attractive support materials in biotrickling filtration.

Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry

2016 ◽  
Vol 2016 (11) ◽  
pp. pdb.prot087288 ◽  
Author(s):  
Lisa C. Crowley ◽  
Brooke J. Marfell ◽  
Adrian P. Scott ◽  
Nigel J. Waterhouse
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Annexin V ◽  
Iodide Uptake ◽  
Apoptosis And Necrosis

scholarly journals The effect of temperature on apoptosis of bovine blood eosinophil granulocytes in vitro

2008 ◽  
Vol 56 (1) ◽  
pp. 173-178 ◽  
Author(s):  
Petr Sláma ◽  
Zbyšek Sládek ◽  
Dušan Ryšánek ◽  
Ivana Burešová
Keyword(s):  
Flow Cytometry ◽  
Uv Irradiation ◽  
Propidium Iodide ◽  
Annexin V ◽  
Positive Control ◽  
Effect Of Temperature ◽  
Blood Eosinophil ◽  
Bovine Blood ◽  
Eosinophil Granulocytes

The aim of the study was to evaluate the effect of temperature on apoptosis of bovine blood eosinophil granulocytes in vitro. Heparinised bovine blood was incubated for 1, 4 and 24 h under following temperatures: 4, 23 and 37 °C. UV irradiation was used as positive control of apoptosis. Eosinophil granu­locytes apoptosis was detected by flow cytometry after simultaneous staining with Annexin-V and propidium iodide. From selected temperatures, 4 °C induced the eosinophil granulocytes apoptosis least. The proportion of apoptotic eosinophil granulocytes amounted to (mean ± SD) 1.65 ± 0.46%; 1.76 ± 0.36%; 4.78 ± 1.70% after 1, 4 and 24 h incubation, respectively.

Omipalisib (GSK458), a Novel Pan-PI3K/mTOR Inhibitor, Exhibits In Vitro Anti-Lymphoma Activity in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5376-5376 ◽  
Author(s):  
Thomas Ippolito ◽  
Greg Tang ◽  
Cory Mavis ◽  
Juan J Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Cell Lines ◽  
Western Blotting ◽  
Propidium Iodide ◽  
Annexin V ◽  
Mtor Pathway ◽  
Pathway Inhibition

Abstract Background: Despite significant gains achieved in the treatment of Burkitt lymphoma (BL), current multi-agent immunochemotherapeutic regimens lead to high rates of acute toxicity, and relapsed/refractory disease still represents a significant hurdle with survival expected in only about 20-30% of such patients. Novel targeted therapeutic approaches are necessary to reduce treatment related toxicity in the up-front setting and improve survival in the relapsed/refractory setting. Analyses of genomic abnormalities in BL have identified increased activation of the PI3K/Akt/mTOR pathway in BL, induced by tonic B-cell receptor signaling and increased expression of Myc induced microRNAs (miRs), as having a significant role in Burkitt lymphomagensis. Additionally, recent reports have implicated higher expression of PI3K activating, Myc induced miRs in pediatric patients with a higher risk of relapse. While focused targeting of PI3K with the PI3K-delta isoform specific inhibitor idelalisib has led to significant activity in indolent B-cell lymphomas, limited activity has been noted in the setting of more aggressive forms. A broader inhibition of both upstream and downstream components of the pathway may exhibit more significant anti-lymphoma activity. To this end, we investigated the in vitro effects of PI3K/Akt/mTOR pathway inhibition with the dual pan-PI3K/mTOR inhibitor Omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models. Methods: The in vitro effect of omipalisib was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell viability following exposure to omipalisib alone and in combination with cytotoxic chemotherapeutic agents was analyzed using Cell-Titer Glo and Alamar Blue assays. Apoptosis was analyzed using western blotting for PARP and by flow cytometry with Annexin V-propidium iodide staining. Downstream targets in the PI3K/Akt/mTOR pathway were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using propidium iodide staining. Synergistic activity of combination exposures was determined by calculation of a combination index using CalcuSyn software. Results: In vitro exposure of BL cell lines to omipalisib in concentrations ranging from 0.05μM to 50μM for 24, 48 or 72 hours resulted in a dose and time dependent decrease in viable cells with significant activity noted at even low nM concentrations (48 hour IC50 values: Raji=1.2μM, Raji 4RH=0.02μM, Raji 8RH=1.9μM, Ramos=0.01μM, Daudi=0.01μM). Flow cytometry for Annexin V and propidium iodide, after 72 hours of single agent exposure to omipalisib, showed a marked induction of apoptosis in all cell lines. For example, at an omipalisib concentration of 200nM, the percentage of Annexin V positive cells were Raji=40.7%, Raji 4RH=4.4%, Raji 8RH=41.5%, Ramos=59.4% and Daudi=46.9%. Approximately ten-fold higher omipalisib concentrations were required to induce similar degrees of apoptosis in the chemotherapy resistant Raji 4RH cell line compared to chemotherapy sensitive cell lines. Western blotting for downstream targets of the PI3K/Akt/mTOR pathway, including S6 and GSK3Β, showed a reduction in phosphorylation after 30 minutes of exposure to omipalisib in all cell lines. Determination of cell cycle progression following exposure to omipalisib for 72 hours at concentrations ranging from 0.006μM to 25μM showed dose-dependent cell cycle arrest in G1 phase in all cell lines; however the chemotherapy resistant Raji 4RH cells arrested in G2/M at higher concentrations. When BL cells were exposed to omipalisib in combination with either doxorubicin or dexamethasone, synergistic anti-tumor activity was observed in all cell lines tested. Conclusion: Inhibition of PI3K and mTOR by the dual inhibitor omipalisib suppresses activation of the PI3K/Akt/mTOR pathway leading to impaired BL cell proliferation with G1 cell cycle arrest and induction of apoptosis in chemotherapy-sensitive and -resistant cell line models of BL. Inhibition of the PI3K/Akt/mTOR pathway with omipalisib also increases the in vitro response to cytotoxic chemotherapeutic agents. Our findings note the pre-clinical activity of PI3K/Akt/mTOR pathway inhibition in BL and highlight the relevance of pursuing PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. Disclosures No relevant conflicts of interest to declare.

Regulated Phosphatidylserine Exposure on Platelets Mediates Fibrin Formation in Hemostasis and Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1645-1645
Author(s):  
Jialan Shi ◽  
Steven W. Pipe ◽  
Jan T. Rasmussen ◽  
Christian W. Heegaard ◽  
Gary E. Gilbert
Keyword(s):  
Flow Cytometry ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Annexin V ◽  
Vascular Wall ◽  
Fibrin Deposition ◽  
Exposure In Vivo

Abstract Thrombin-stimulated platelets support activity of phosphatidylserine (PS)-dependent blood coagulation reactions. However, only 2–6% of stimulated platelets expose sufficient PS to bind annexin V, leading to the supposition that procoagulant reactions are localized to the annexin V positive platelets and to microparticles shed by platelets. We hypothesized that thrombin-stimulated platelets expose sufficient PS to support the prothrombinase and factor Xase complexes but insufficient to meet the threshold for annexin V binding. We evaluated lactadherin, a PS-binding milk protein, as a reagent to detect platelet PS exposure. Thrombin or TRAP-treated platelets bound lactadherin with 3200 ± 700 sites/plt, but did not bind annexin V, as detected by flow cytometry. To confirm that lactadherin binding truly reported PS exposure, we performed activation experiments upon platelets loaded with 1-Palmitoyl-2- [6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-Glycero-3-Phospho-L-Serine (NBD-PS). Stimulation of platelets with 10 μM TRAP led to 8 ± 2 % PS exposure vs > 90% PS exposure for 10 μM A23187. PS exposure was maximal within 1 min of exposure to TRAP and was reversible with >70% of exposed PS re-internalized within 30 min. In situ platelet fibrin deposition was monitored utilizing a novel flow cytometry assay. Platelet rich plasma was recalcified and supplemented with 50 pM factor Xa, 100 μM GPRP. Platelet-bound fibrin, lactadherin and annexin V binding sites were measured at time intervals. Fibrin accumulated on lactadherin + platelets at a rate greater than or equal to the rate on platelet microparticles or annexin V + platelets. Lactadherin inhibited > 98% of prothrombinase and factor Xase activity on platelets while annexin V inhibition reached a plateau of ~ 80%. The localization and importance of regulated PS exposure in vivo was evaluated in mouse models. Anesthetized mice were injected intravenously with 1 μg of lactadherin and 1 μg annexin V. Three minutes after FeCl3 injury of exposed mesentery, mice were perfused with fixative. Immunohistochemistry showed veins edged with fibrin and decorated with platelets. Lactadherin co-localized with CD41+ platelets along the vascular wall but was less intense on platelets lodged within fibrin aggregates that extended into the vessel lumen and did not stain platelets in sequestered blood. Annexin V stained only scattered platelets and endothelial cells and did not correlate well to sites of fibrin deposition. To evaluate the hemostatic importance of platelet PS, 10 μg of lactadherin was injected intravenously prior to tail snip; bleeding volumes increased from 33 ± 29 μl to 128 ± 39 μl, n experimental = 8. Rose bengal/laser-induced carotid thrombosis delayed from 34 ± 9 min to 80 ± 20 min, n = 10 after 8–21 μg lactadherin. Four treated animals did not develop a thrombosis. In summary, use of lactadherin as a PS probe has revealed that the capacity for regulated PS exposure, at levels below the annexin V binding threshold, is a general platelet property and that low level PS exposure is sufficient to support thrombin and fibrin generation in vitro and in vivo. These results have implications about the mechanism through which PS exposure is regulated as well as for the potential value of PS exposure as a diagnostic or therapeutic target.

scholarly journals P11.60 Neurosurgical delivery of the poly ADP ribose polymerase-1 inhibitor olaparib from a thermo-responsive biodegradable paste potentiates radiotherapy and prolongs survival

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii57-iii57
Author(s):  
S Smith ◽  
R Serra ◽  
J Rowlinson ◽  
N Gorelick ◽  
G Veal ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Adjuvant Radiotherapy ◽  
Clonal Growth ◽  
Survival Benefit ◽  
Glioma Cells ◽  
Annexin V ◽  
Standard Of Care ◽  
High Grade Glioma ◽  
High Grade

Abstract BACKGROUND There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance. Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed PLGA/PEG thermo-sensitive biodegradable paste. MATERIAL AND METHODS Metabolic and clonogenic assays were used to assess effects on proliferation and clonal growth upon in vitro glioma exposure to olaparib. Flow cytometry and Annexin V/Propidium iodide were used to determine apoptosis. The 9L high-grade glioma orthotopic allograft model was utilised to assess survival upon intra-cavity olaparib delivery. RESULTS Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study revealed a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (95 vs. 44 vs. 30 days respectively). A more pronounced survival benefit, as measured by number of animals surviving long-term, was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy, relative to standard-of-care (95 vs. 44 days). Clinical correlation was determined using RNAseq data from 10 GBM patients, showing significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence-activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells. CONCLUSION Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.

scholarly journals On the Origin of Tetraploid Vernal Grasses (Anthoxanthum) in Europe

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 966
Author(s):  
Zuzana Chumová ◽  
Terezie Mandáková ◽  
Pavel Trávníček
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Crucial Role ◽  
Evolutionary History ◽  
Essential Role ◽  
Grass Species ◽  
Polyploid Species ◽  
Anthoxanthum Odoratum ◽  
European Populations

Polyploidy has played a crucial role in the evolution of many plant taxa, namely in higher latitudinal zones. Surprisingly, after several decades of an intensive research on polyploids, there are still common polyploid species whose evolutionary history is virtually unknown. Here, we addressed the origin of sweet vernal grass (Anthoxanthum odoratum) using flow cytometry, DNA sequencing, and in situ hybridization-based cytogenetic techniques. An allotetraploid and polytopic origin of the species has been verified. The chromosome study reveals an extensive variation between the European populations. In contrast, an autopolyploid origin of the rarer tetraploid vernal grass species, A. alpinum, has been corroborated. Diploid A. alpinum played an essential role in the polyploidization of both European tetraploids studied.

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