scholarly journals Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

10.3791/2597 ◽  
2011 ◽  
Author(s):  
Aja M. Rieger ◽  
Kimberly L. Nelson ◽  
Jeffrey D. Konowalchuk ◽  
Daniel R. Barreda
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
Annexin V ◽  
Accurate Assessment ◽  
Apoptosis Assay

Type II secretory phospholipase A2 binds to ischemic flip-flopped cardiomyocytes and subsequently induces cell death

2003 ◽  
Vol 285 (5) ◽  
pp. H2218-H2224 ◽  
Author(s):  
R. Nijmeijer ◽  
M. Willemsen ◽  
C. J. L. M. Meijer ◽  
C. A. Visser ◽  
R. H. Verheijen ◽  
...  
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
Caspase 3 ◽  
Ischemia Reperfusion ◽  
Annexin V ◽  
Border Zone ◽  
Metabolic Inhibitors ◽  
Type Ii ◽  
Secretory Phospholipase

Type II secretory phospholipase A2 (sPLA2) is a cardiovascular risk factor. We recently found depositions of sPLA2 in the necrotic center of infarcted human myocardium and normally appearing cardiomyocytes adjacent to the border zone. The consequences of binding of sPLA2 to ischemic cardiomyocytes are not known. To explore a potential effect of sPLA2 on ischemic cardiomyocytes at a cellular level we used an in vitro model. The cardiomyocyte cell line H9c2 or adult cardiomyocytes were isolated from rabbits that were incubated with sPLA2 in the presence of metabolic inhibitors to mimic ischemia-reperfusion conditions. Cell viability was established with the use of annexin V and propidium iodide or 7-aminoactinomycin D. Metabolic inhibition induced an increase of the number of flip-flopped cells, including a population that did not stain with propidium iodide and that was caspase-3 negative. sPLA2 bound to the flip-flopped cells, including those negative for caspase-3. sPLA2 binding induced cell death in these latter cells. In addition, sPLA2 potentiated the binding of C-reactive protein (CRP) to these cells. We conclude that by binding to flip-flopped cardiomyocytes, including those that are caspase-3 negative and presumably reversibly injured, sPLA2 may induce cell death and tag these cells with CRP.

9-Methoxycamptothecin from Nothapodytes foetida Induces Apoptosis in Murine Sarcoma S180 Cells

2011 ◽  
Vol 66 (9-10) ◽  
pp. 471-476
Author(s):  
Na Liao ◽  
Peng Zhang ◽  
Mingzhang Ao ◽  
Jing Wang ◽  
Yueyuan Shi ◽  
...  
Keyword(s):  
Cell Death ◽  
Nmr Spectroscopy ◽  
Mtt Assay ◽  
Propidium Iodide ◽  
Annexin V ◽  
Double Staining ◽  
Nothapodytes Foetida ◽  
Ic50 Value ◽  
Mitochondria Pathway ◽  
Murine Sarcoma

9-Methoxycamptothecin (MCPT) was found to have antitumour activities through topoisomerase inhibition. However, the type of cell death induced in the tumour cells treated with MCPT was not elucidated. In this study, MCPT and camptothecin were isolated from Nothapodytes foetida distributed in Hubei Province, China and identifi ed by NMR spectroscopy. MCPT was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay using camptothecin as reference. Annexin V-FITC/propidium iodide double staining and real-time PCR were also performed. The IC50 value was (0.385 ± 0.08) μM. The apoptosis rates increased from 9.5% to 17.27%, 30.14%, and 66.46% with an increase in MCPT concentrations from 0, 0.19, 0.38, to 0.95 μM, respectively. The ratio of Bax/Bcl-2 also increased from 1 to 1.61, 2.43, and 4.57, respectively. Bax and Bcl-2 are crucial to the mitochondria pathway. The results indicate that the mitochondria pathway may be involed in MCPT-induced murine sarcoma S180 apoptosis.

scholarly journals Zerumbone inhibits prostate cancer cell viability and induces cell death by non-apoptotic pathway

2016 ◽  
Vol 11 (4) ◽  
pp. 771
Author(s):  
Xian-De Cao ◽  
Hui-Min Zheng
Keyword(s):  
Prostate Cancer ◽  
Cell Death ◽  
Cell Viability ◽  
Cancer Cell ◽  
Propidium Iodide ◽  
Prostate Cancer Cell ◽  
Apoptotic Cell ◽  
D1 Protein ◽  
Annexin V ◽  
Apoptotic Pathway

<p class="Abstract">The aim of the present study was to investigate the role of zerumbone on the proliferation, cell cycle arrest and cell death in DU-145 prostate cancer cell lines. The MTT assay revealed that zerumbone (20 µM) reduced proliferation of DU-145 cells to 39.0% at 48 hours. It also increased the proportion of propidium iodide stained cells to 53.4% compared 1.0% in control. However, the population of annexin V-stained cells remained uneffected indicating induction of non-apoptotic cell death by zerumbone. Treatment of DU-145 cells with zerumbone (20 µM) caused 8-fold enhancement in the level of reactive oxygen species (ROS). On the other hand, exposure of the zerumbone treated DU-145 cells to glutathione inhibited the generation of ROS. Fow cytometry using propidium iodide staining revealed that zerumbone treat-ment increased proportion of cells in G1 phase to 71.3% on compared to 34.7% in the control. The results from Western blot analysis revealed a significant increase in the expression of cyclin D1 protein in DU-145 cells on treatment with 20 µM concentration of zerumbone. Thus, zerumbone treatment inhibits prostate cancer cell viability and can be used for its treatment.</p><p> </p>

Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay v1

2021 ◽  
Author(s):  
Catherine M. Worsley ◽  
Rob B. Veale ◽  
Elizabeth S. Mayne
Keyword(s):  
Cell Death ◽  
Chemical Treatment ◽  
Structural Damage ◽  
Viral Infections ◽  
Annexin V ◽  
Laboratory Methods ◽  
Cellular Processes ◽  
Flow Cytometric ◽  
Apoptosis Assay ◽  
Tumour Cell Lines

Cell death is a key component of mammalian physiology, and can happen as a result of structural damage, or actively as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V apoptosis assay.

scholarly journals Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland neutrophils in vitro

2012 ◽  
Vol 50 (No. 1) ◽  
pp. 11-23 ◽  
Author(s):  
Z. Sladek ◽  
D. Rysanek ◽  
M. Faldyna
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Death ◽  
Mammary Gland ◽  
Programmed Cell Death ◽  
Propidium Iodide ◽  
Annexin V ◽  
Dna Fragments ◽  
Bovine Mammary Gland ◽  
Streptococcus Uberis

Neutrophils play an important role in the defence of the bovine mammary gland against bacterial infections. In the course of the resolution of mammary gland inflammation, neutrophils undergo programmed cell death &ndash; apoptosis. The aim of this study was to confirm whether the co-cultivation of neutrophils of the bovine mammary gland with either Staphylococcus aureus or Streptococcus uberis leads to signs of apoptosis. In the study, 16 mammary glands of four virgin heifers aged 16 to 18 months were examined. Neutrophils were obtained by lavage after an induced influx. After a three-hour incubation of the neutrophils with bacteria in vitro, neutrophil apoptosis was detected by morphological features, by determination of histone-associated DNA fragments (ELISA), and by Annexin -V and propidium iodide positivity (flow cytometry). S. aureus and S. uberis reduced the incidence of karyopycnotic and zeiotic neutrophils (P &lt; 0.01), and insignificantly reduced the concentration of histone -associated DNA fragments (P &gt; 0.05). The incubation of neutrophils with bacteria, however, increased the proportion of Annexin &ndash;V-positive cells (P &lt; 0.01) and Annexin -V and propidium iodide-positive cells (P &lt; 0.05). Co-cultivation of neutrophils with either S. aureus or S. uberis led to the induction of phosphatidylserine translocation characteristic of the early stage of apoptosis. The late signs of apoptosis were delayed by co-cultivation of neutrophils with both pathogens. Therefore it is obvious that although the programmed cell death of apoptosis is initiated by these pathogens, the completion of the program is delayed.

Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death

2010 ◽  
Vol 358 (1-2) ◽  
pp. 81-92 ◽  
Author(s):  
Aja M. Rieger ◽  
Brian E. Hall ◽  
Le Thuong Luong ◽  
Luis M. Schang ◽  
Daniel R. Barreda
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
False Positives ◽  
Accurate Assessment

scholarly journals Changes in cell death of peripheral blood lymphocytes isolated from children with acute lymphoblastic leukemia upon stimulation with 7 Hz, 30 mT pulsed electromagnetic field

2015 ◽  
Vol 20 (1) ◽  
Author(s):  
Jolanta Kaszuba-Zwoińska ◽  
Magdalena Ćwiklińska ◽  
Walentyna Balwierz ◽  
Paulina Chorobik ◽  
Bernadeta Nowak ◽  
...  
Keyword(s):  
Cell Death ◽  
Electromagnetic Field ◽  
Acute Lymphoblastic Leukemia ◽  
Propidium Iodide ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Pulsed Electromagnetic Field ◽  
Pulsed Electromagnetic ◽  
In Cells

AbstractPulsed electromagnetic field (PEMF) influenced the viability of proliferating in vitro peripheral blood mononuclear cells (PBMCs) isolated from Crohn’s disease patients as well as acute myeloblastic leukemia (AML) patients by induction of cell death, but did not cause any vital changes in cells from healthy donors. Experiments with lymphoid U937 and monocytic MonoMac6 cell lines have shown a protective effect of PEMF on the death process in cells treated with death inducers.The aim of the current study was to investigate the influence of PEMF on native proliferating leukocytes originating from newly diagnosed acute lymphoblastic leukemia (ALL) patients.The effects of exposure to PEMF were studied in PBMCs from 20 children with ALL. PBMCs were stimulated with three doses of PEMF (7 Hz, 30 mT) for 4 h each with 24 h intervals. After the last stimulation, the cells were double stained with annexin V and propidium iodide dye to estimate viability by flow cytometric analysis.The results indicated an increase of annexin V positive as well as double stained annexin V and propidium iodide positive cells after exposure to threefold PEMF stimulation.A low-frequency pulsed electromagnetic field induces cell death in native proliferating cells isolated from ALL patients. The increased vulnerability of proliferating PBMCs to PEMF-induced interactions may be potentially applied in the therapy of ALL.The analysis of expression of apoptosis-related genes revealed changes in mRNA of some genes engaged in the intrinsic apoptotic pathway belonging to the Bcl-2 family and the pathway with apoptosis-inducing factor (AIF) abundance upon PEMF stimulation of PBMCs.

B Cells Are Primed for Survival by BAFF-Driven Bim Degradation In Chronic Graft Versus Host Disease (cGVHD) Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 216-216
Author(s):  
Jenna Wooten ◽  
Bhuiya S. Nazmim ◽  
Kristy Richards ◽  
Andrew Sharf ◽  
Joseph H. Antin ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Propidium Iodide ◽  
Ex Vivo ◽  
Annexin V ◽  
Healthy Individuals ◽  
Graft Versus Host ◽  
Bh3 Mimetic ◽  
Activated B Cells

Abstract Abstract 216 High BAFF levels correlate with the presence of activated B cells in patients who develop cGVHD after hematopoietic stem cell transplantation. B cell reconstitution in these patients occurs under constant exposure to alloantigens, and we previously showed that B Cell Receptor (BCR) stimulated CD27+ B cells in cGVHD patients are activated, capable of spontaneous IgG production without requirement of further BCR or second signal stimulation. B cell survival is dependent on both BCR and BAFF signaling. BAFF is known to attenuate B cell apoptosis by counteracting pro-apoptotic Bcl-2-interacting mediator of cell death (Bim) protein, but it is not known whether BAFF can provide survival signals to activated B cells in cGVHD. Therefore, we examined the survival rates of B cells in cGVHD. CD19+ B cells were purified (>95% purity) by magnetic bead sorting. Rates of death were measured by flow cytometry staining with propidium iodide and Annexin V of unmanipulated B cells cultured without addition of cytokines over time. After 24 and 48 hours, the frequency of cells undergoing apoptosis (Annexin V+) B cells was significantly lower in samples from patients with cGVHD compared to those without cGVHD and to healthy individuals (one-way ANOVA p=0.007, Figure 1). In addition to Annexin V + cells, total death rates as measured by propidium iodide of unmanipulated purified CD27+ B cells were lower in cGVHD compared to healthy individuals. Importantly, we found that the frequency of propidium iodide stained CD27+ B cells did not increase 24 hours ex vivo if BAFF was added in cGVHD, but not in healthy, CD27+ B cells, consistent with BAFF mediated survival in these cells (Table 1). Further examination of CD27+ B cell subsets ex vivo was performed to determine if subpopulations we previously identified to uniquely circulate in cGVHD patients were more viable. The morphology of pre-germinal center (GC) CD27+IgD+CD38Hi cells and the antigen-inexperienced, most recent bone marrow emigrants, transitional CD27NegIgD+CD38Hi cells was compared. Unlike transitional cells, the pre-GC cells were enlarged, adherent and viable, consistent with an activated state. While the Bim isoforms are upregulated after BCR activation or by apoptosis-inducing drugs, Bim is degraded in response to BAFF signaling. Since steroids (previously shown to increase Bim in lymphocytes) are the only standard therapy for cGVHD and unfortunately often clinically ineffective, we first performed in vitro assays with dexamethasone and BAFF. Ninety-five percent of healthy CD19+ purified B cells were induced to apoptose (94.9% Annexin V+) with dexamathasone at 24 hours. Addition of BAFF blocked dexamethasone-induced apoptosis to the baseline levels found in untreated B cells (27.3% Annexin V+). Next, to determine whether in vivo BAFF survival signaling of B cells occurred in cGVHD patients, we examined protein levels of Bim by immunoblotting cell lysates from freshly purified unmanipulated CD19+ cells. B cells from healthy individuals did not generate the long form of Bim (BimL), likely due to the lack of BCR activation in these B cells, while 80% of patients with inactive cGVHD had increased BimL. In contrast, 75% of B cells from patients with active cGVHD lacked BimL. Thus, loss of BimL in cGVHD is likely BAFF-driven and may contribute to improved survival of potentially allo- or autoreactive B cells in cGVHD. Potential upstream activators of Bim degradation and inhibition such as mitogen-activated protein kinase/ERK activating kinase (MEK) or NFkB, respectively, are currently being investigated. In addition to characterizing a potential therapeutic role for MEK and NFkB inhibitors, since Bim has been shown to be increased with proteasome inhibitor and BH3 mimetic induced cell death, these findings begin to delineate immunologic rationale for the therapeutic use of these agents to target B cells in cGVHD. Taken together, our data suggest that activated and potentially pathologic B cells in cGVHD utilize distinct survival pathways. Thus, activated B cells represent novel therapeutic targets in cGVHD.Table 1.CD27+ B Cell Source% PI at Time 0 (mean +/-SD)% PI at 24 Hours (mean +/-SD)% PI at 48 Hours (mean +/-SD)Healthy12.8 +/- 10.7 (n=3)34.9 +/- 8.7 (n=2)34.3 +/- 9.6 (n=3)Healthy +BAFF33.1 +/- 9.1 (n=2)42.2 +/- 11.4 (n=2)Yes cGVHD13.5 +/- 5.9 (n=3)20.5 +/- 3.1 (n=2)34.8 +/- 4.0 (n=3)Yes cGVHD +BAFF14.8 +/- 5.4 (n=2)29.7 +/- 2.7 (n=2) Disclosures: Off Label Use: NFkB inhibitor, MEK inhibitor, BH3 mimetic, bortezomib for chronic graft versus host disease.

scholarly journals Relationship between Virus Replication and Apoptosis Events in IgM + Cells from Chicken Spleen and Bursa of Fabricius Infected with Malaysia Strain of Very Virulent Infectious Bursal Disease Virus

2018 ◽  
Vol 44 (1) ◽  
pp. 9 ◽  
Author(s):  
Kristeen Ye Wen Teo ◽  
Yeap Swee Keong ◽  
Tan Sheau Wei ◽  
Abdul Rahman Omar ◽  
Noorjahan Banu Alitheen
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Cell Death ◽  
Cell Viability ◽  
B Lymphocytes ◽  
Propidium Iodide ◽  
Annexin V ◽  
Bursa Of Fabricius ◽  
Chicken Spleen ◽  
Double Stain

Background: Infection of IBDV was reported to be endemic in worldwide including Malaysia and can be spread orally thru polluted fodder and water source, thus causing economic losses especially in commercial poultry industry. The infection resulted in depletion of B lymphocytes and subsequently destruction of the bursa which leaded to immunosuppression of the bird and it was postulated that the depletion of cells in the bursa was due to induction of apoptosis. In the current study, the infection of Malaysia isolated very virulent IBDV UPM0081 on IgM bearing B lymphocytes (IgM+ cells) from chicken spleen and bursa was compared.Materials, Methods & Results: A total of sixty eggs were obtained and raised until the age of 3 weeks old. The birds were divided into two groups (n = 30), which one of them served as control while IBDV strain UPM0081 was used to infect another group of birds at the concentration of 103 ELD50. The birds were observed and sacrificed at day 2, 4 and 5 post infections. Spleen and bursa of Fabricius were harvested and subjected to IgM+ cell enrichment using microbeads. The cell viability of enriched cells was assayed using MTT and cell cycle was analyzed using propidium iodide. Annexin V FITC and acridine orange/propidium iodide double stain assays were used to determine the event of apoptosis in the enriched IgM+ cells. Also, the IBDV viral load was also quantified by using real time PCR to evaluate the relationship between virus replication and apoptosis events in the infected chickens. Current results showed that the apoptotic events were observed to be significantly higher in IgM+ cells isolated from chicken bursa as compared to the cells isolated from spleen. The bursal B lymphocytes cell viability was observed to be decreasing following the infection of very virulent IBDV. The cells were then investigated of their apoptotic rate and data showed that increasing apoptotic cells (early and late apoptosis) were observed in AO/PI double stain as well as increment of SubG0/G1 population in the cell cycle analysis and also increment of Annexin V FITC bound cells in the apoptosis study. As for B lymphocytes from chicken spleen, the magnitude of damage caused by very virulent IBDV was not as severe as what being observed in the chicken bursa, with the cell viability drastically decreased on day 4 following IBDV infection.Discussion: IBDV caused severe destruction in bursa of Fabricius compared to spleen, in which cell death events in the former was reported to be directly caused by the virus. Apoptotic event in chicken spleen following IBDV infection was observed to be caused by oxidative stress. Thus, viral replication played a role in inducing bursal IgM+ cells death while such phenomenon was not observed in spleen isolated IgM+ cells. In summary, the cell death events of IgM+ cells in chicken spleen and bursa of Fabricius may be accounted by different factors upon infection with Malaysia strain of IBDV UPM0081. It is obvious that IgM+ cells from chicken bursa suffered from apoptotic cell death in an increasing manner considerably with time of infection and RNA load detected in the cells, which supported by previous literature that IBDV induces host cells apoptosis, with both VP2 and VP5 playing a role in binding and apoptosis. Meanwhile, the cell death events of B lymphocytes in chicken spleen was observed to be more relevant to other factors such as the oxidative stress or proinflammatory cytokines that caused by the virus infection rather than the viral RNA load.

scholarly journals Action of inhibitors Heat shock proteins 90 and 27 on dexamethasone-induced apoptosis of tumor cells

2010 ◽  
Vol 9 (3) ◽  
pp. 68-71 ◽  
Author(s):  
Ye. V. Kaigorodova ◽  
N. V. Ryazantseva ◽  
V. V. Novitsky ◽  
A. N. Maroshkina ◽  
M. V. Belkina ◽  
...  
Keyword(s):  
Cell Death ◽  
Heat Shock ◽  
Programmed Cell Death ◽  
Heat Shock Protein ◽  
Tumor Cells ◽  
Propidium Iodide ◽  
Fluorescent Microscopy ◽  
Annexin V ◽  
Induced Apoptosis ◽  
Shock Proteins

Programmed cell death of tumor cells of line Jurkat in conditions of cultivation with various concentration of dexamethasone, selective inhibitors of Hsp90 (Heat shock protein — Hsp) (17-AAG) and Hsp27 (KRIBB3) was investigated. An estimation of realisation apoptosis spent by method of fluorescent microscopy with use annexin V-FITC and propidium iodide. Inhibition of Hsp90 and Hsp27 leads to activation of tumor cells Jurkat apoptotic program and strengthening of dexamethasone-induced apoptosis. Hsp27 and Hsр90 play an antiapoptotic role in tumor cells of line Jurkat.

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