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

Annexin V staining during reperfusion detects cardiomyocytes with unique properties

2001 ◽  
Vol 281 (5) ◽  
pp. H1931-H1937 ◽  
Author(s):  
Prakash Narayan ◽  
Robert M. Mentzer ◽  
Robert D. Lasley
Keyword(s):  
Propidium Iodide ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Annexin V ◽  
Cell Width ◽  
Simulated Ischemia ◽  
Propidium Iodide Staining ◽  
Membrane Blebs ◽  
Apoptosis And Necrosis

With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8–10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+(111 ± 14 nM) was elevated in reperfused annexin V-negative cells (214 ± 22 nM), and further elevated in annexin V-positive myocytes (382 ± 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in ∼3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.

scholarly journals Critical Evaluation of Techniques to Detect and Measure Cell Death – Study in a Model of UV Radiation of the Leukaemic Cell Line HL60

1999 ◽  
Vol 19 (3-4) ◽  
pp. 139-151 ◽  
Author(s):  
Marina Leite ◽  
Margarida Quinta‐Costa ◽  
Pedro Simas Leite ◽  
José Eduardo Guimarães
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Fluorescence Microscopy ◽  
Trypan Blue ◽  
Critical Evaluation ◽  
Annexin V ◽  
Tunel Assay ◽  
Apoptotic Cells ◽  
Dna Ladder ◽  
Apoptosis And Necrosis

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V‐FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.

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.

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.

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 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.

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