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 Cytolethal Distending Toxin Induces Caspase-Dependent and -Independent Cell Death in MOLT-4 Cells

2008 ◽  
Vol 76 (10) ◽  
pp. 4783-4791 ◽  
Author(s):  
Masaru Ohara ◽  
Tomonori Hayashi ◽  
Yoichiro Kusunoki ◽  
Kei Nakachi ◽  
Tamaki Fujiwara ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Population ◽  
Apoptotic Cell ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Membrane Disruption ◽  
Classical Pathway ◽  
Cytolethal Distending Toxin ◽  
Flow Cytometric ◽  
General Caspase Inhibitor

ABSTRACT Cytolethal distending toxin (CDT) induces apoptosis using the caspase-dependent classical pathway in the majority of human leukemic T cells (MOLT-4). However, we found the process to cell death is only partially inhibited by pretreatment of the cells with a general caspase inhibitor, z-VAD-fmk. Flow cytometric analysis using annexin V and propidium iodide showed that a 48-h CDT treatment decreased the living cell population by 35% even in the presence of z-VAD-fmk. z-VAD-fmk completely inhibited caspase activity in 24 h CDT-intoxicated cells. Further, CDT with z-VAD-fmk treatment clearly increased the cell population that had a low level of intracellular reactive oxygen. This is a characteristic opposite to that of caspase-dependent apoptosis. Overexpression of bcl2 almost completely inhibited cell death using CDT treatment in the presence of z-VAD-fmk. The data suggest there are at least two different pathways used in CDT-induced cell death: conventional caspase-dependent (early) apoptotic cell death and caspase-independent (late) death. Both occur via the mitochondrial membrane disruption pathway.

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

vitreal cells and specialized phagocytes in the chick embryo retina: A TEM and SEM study

1990 ◽  
Vol 48 (3) ◽  
pp. 330-331
Author(s):  
M.A. Cuadros ◽  
M.J. Martinez-Guerrero ◽  
A. Rios
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Chick Embryo ◽  
Basement Membrane ◽  
Sem Images ◽  
Intimate Contact ◽  
Cellular Processes ◽  
Sem Study ◽  
Free Cells

In the chick embryo retina (days 3-4 of incubation), coinciding with an increase in cell death, specialized phagocytes characterized by intense acid phosphatase activity have been described. In these preparations, all free cells in the vitreal humor (vitreal cells) were strongly labeled. Conventional TEM and SEM techniques were used to characterize them and attempt to determine their relationship with retinal phagocytes.Two types of vitreal cells were distinguished. The first are located at some distance from the basement membrane of the neuroepithelium, and are rounded, with numerous vacuoles and thin cytoplasmic prolongations. Images of exo- and or endocytosis were frequent; the cells showed a well-developed Golgi apparatus (Fig. 1) In SEM images, the cells was covered with short cellular processes (Fig. 3). Cells lying parallel to or alongside the basement membrane are elongated. The plasma membrane is frequently in intimate contact with the basement membrane. These cells have generally a large cytoplasmic expansion (Fig. 5).

Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death

2019 ◽  
Vol 18 (10) ◽  
pp. 1448-1456 ◽  
Author(s):  
Bahareh Movafegh ◽  
Razieh Jalal ◽  
Zobeideh Mohammadi ◽  
Seyyede A. Aldaghi
Keyword(s):  
Cell Death ◽  
Cellular Uptake ◽  
Combined Treatment ◽  
Annexin V ◽  
Cell Penetrating Peptides ◽  
Short Exposure ◽  
Dependent Manner ◽  
Du145 Cells ◽  
Induced Apoptosis ◽  
Resistance To Doxorubicin

Objective: Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell-penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. Methods: The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide- acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicininduced cell death. Results: Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24h combined treatment of cells with doxorubicin (0.5 µM) and poly-L-arginine (1 µg ml-1) caused a small increase in doxorubicin-induced apoptosis and significantly elevated necrosis in DU145 cells as compared to each agent alone. Conclusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferationinducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis.

scholarly journals Hydrogen-Generating Silica Material Prevents UVA-Ray-Induced Cellular Oxidative Stress, Cell Death, Collagen Loss and Melanogenesis in Human Cells and 3D Skin Equivalents

Antioxidants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 76
Author(s):  
Li Xiao ◽  
Mai Mochizuki ◽  
Taka Nakahara ◽  
Nobuhiko Miwa
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Annexin V ◽  
Human Keratinocytes ◽  
Absorption Capacity ◽  
Human Cells ◽  
Skin Equivalents ◽  
Type Iv ◽  
Silica Material ◽  
3D Skin

Ultraviolet-A (UVA) irradiation induces harmful effects on skin cells and accelerates skin aging through oxidative stress. In this study, the effects of a hydrogen-generating silica material named ULH-002 against UVA injuries in human cells and 3D skin equivalents were investigated. The oxygen radical absorption capacity (ORAC) assay showed that both freshly prepared ULH-002 solutions and 7-day-old solutions exhibited equal peroxyl radical (ROO·) scavenging activities concentration-dependently. CellROX® green/orange staining showed that ULH-002 could reduce UVA-induced oxidative stress in human keratinocytes HaCaT and human gingival fibroblasts (HGFs). ULH-002 significantly prevented UVA-induced apoptotic/necrotic cell death and cell-viability decline in HGFs and keratinocytes, as shown by Annexin V/PI apoptosis assay and PrestoBlue assay, respectively. Immunostaining showed that ULH-002 prevented the UVA-induced deterioration of expression of both type IV and I collagens in the 3D skin equivalents, and similarly in monolayer HGFs. UVA-enhanced melanogenesis was observed in human melanocytes HMV-II and HMV-II cell-containing 3D skin equivalents, but markedly prevented by ULH-002 as demonstrated by Fontana–Masson’s staining. In conclusion, our data suggested that ULH-002 could protect human keratinocytes and fibroblasts from UVA-induced injuries, prevent the loss of type IV and I collagens, as well as reduce melanogenesis. ULH-002 might be developed as a skin care reagent in the cosmetic industry.

scholarly journals Cell Death in Coronavirus Infections: Uncovering Its Role during COVID-19

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1585
Author(s):  
Annamaria Paolini ◽  
Rebecca Borella ◽  
Sara De Biasi ◽  
Anita Neroni ◽  
Marco Mattioli ◽  
...  
Keyword(s):  
Cell Death ◽  
Immune Cells ◽  
Viral Infections ◽  
Virus Entry ◽  
Therapeutic Strategies ◽  
The Other ◽  
Cytokine Storm ◽  
Cytokines And Chemokines ◽  
Cell Death Mechanisms ◽  
The One

Cell death mechanisms are crucial to maintain an appropriate environment for the functionality of healthy cells. However, during viral infections, dysregulation of these processes can be present and can participate in the pathogenetic mechanisms of the disease. In this review, we describe some features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and some immunopathogenic mechanisms characterizing the present coronavirus disease (COVID-19). Lymphopenia and monocytopenia are important contributors to COVID-19 immunopathogenesis. The fine mechanisms underlying these phenomena are still unknown, and several hypotheses have been raised, some of which assign a role to cell death as far as the reduction of specific types of immune cells is concerned. Thus, we discuss three major pathways such as apoptosis, necroptosis, and pyroptosis, and suggest that all of them likely occur simultaneously in COVID-19 patients. We describe that SARS-CoV-2 can have both a direct and an indirect role in inducing cell death. Indeed, on the one hand, cell death can be caused by the virus entry into cells, on the other, the excessive concentration of cytokines and chemokines, a process that is known as a COVID-19-related cytokine storm, exerts deleterious effects on circulating immune cells. However, the overall knowledge of these mechanisms is still scarce and further studies are needed to delineate new therapeutic strategies.

The Clinical Significance of miR-135b-5p and Its Role in the Proliferation and Apoptosis of Hippocampus Neurons in Children with Temporal Lobe Epilepsy

2020 ◽  
Vol 42 (5-6) ◽  
pp. 187-194
Author(s):  
Ruixiang Li ◽  
Jiahua Hu ◽  
Sue Cao
Keyword(s):  
Temporal Lobe Epilepsy ◽  
Cell Viability ◽  
Temporal Lobe ◽  
Luciferase Reporter ◽  
Diagnostic Biomarker ◽  
Flow Cytometric ◽  
Proliferation And Apoptosis ◽  
Apoptosis Assay ◽  
Polymerase Chain ◽  
Adverse Influence

Temporal lobe epilepsy (TLE) is the most familiar localized epilepsy in children. MicroRNAs (miRNAs) are essential for the inhibition or promotion of numerous diseases. This study aimed to detect the expression of miR-135b-5p and primarily uncover its underlying function and mechanism in children with TLE. Quantitative real-time polymerase chain reaction was used to evaluate the expression of miR-135b-5p in children with TLE and in a rat model of epilepsy. MTT assay and flow cytometric apoptosis assay were conducted to evaluate the effects of miR-135b-5p on cell viability and apoptosis. Additionally, the dual luciferase reporter assay was performed to confirm the direct target of miR-135b-5p. Our data showed that the expression of miR-135b-5p was significantly decreased in children with TLE and in the epileptic rat neuron model. The dysregulation of miR-135b-5p could serve as a promising diagnostic biomarker for children with TLE. The overexpression of miR-135b-5p moderated the adverse influence on cell viability and apoptosis induced by magnesium-free medium. SIRT1 was identified as a target gene of miR-135b-5p. These results proved that miR-135b-5p might serve as a potential diagnostic biomarker in children with TLE. Overexpression of miR-135b-5p alleviates the postepileptic influence on cell viability and apoptosis by targeting SIRT1.

IMMUNOLOGICAL DISORDERS OF THE REPRODUCTIVE SYSTEM THAT OCCUR WHEN EXPOSED TO BENZENE, IN EMPLOYEES OF OIL-PRODUCING ENTERPRISES

2021 ◽  
Author(s):  
I.V. Leshkova ◽  
◽  
O.V. Dolgih ◽  
O.YU. Ustinova
Keyword(s):  
Cell Death ◽  
Reproductive Health ◽  
Reproductive System ◽  
Death Receptor ◽  
Annexin V ◽  
Reference Level ◽  
Immunological Disorders ◽  
Cellular Expression ◽  
Cell Death Receptor

Abstract. Introduction. The protection of the reproductive health of the working-age population is the most important direction of State policy. In 5-15% of cases, the causes of reproductive dysfunction are immunological disorders. Benzene belongs to the group of industrial reprotoxicants, however, its effect of benzene on the reproductive system has not been sufficiently studied. Objective: to study the immunological aspects of the effect of benzene on the reproductive system. Methods. We examined 50 men exposed to benzene with reproductive disorders (26-49 years old), as well as 4 workers with normal sexual function aged 53-60 years. Spontaneous and induced changes in the cellular expression of apoptosis markers were studied. For the study, the ANNEXIN V-FITC/7-AAD kit was used for the detection of cells that have undergone apoptosis. The experiment was conducted in vitro using a biological medium (ejaculate). A factor of the chemical nature was benzene. Results. According to the results of the comparative analysis, there were no significant deviations of pathogenetic tests of immunological markers in comparison with the reference level in the spontaneous expression samples, but there was an excess of expression of the CD95 + cell death receptor (p<0.05) in 30% of the samples examined, and a decrease in the number of Annexin V-FITC+7AAD negative cells (without reaching the significance level) in samples with a load of (15%). There was a difference in the expression levels of CD95+ and CD25+ CD-reception indicators by 20% and 10% in relation to the spontaneous level (p<0.05). Representatives of the chemical group of aromatic hydrocarbons realize reprotoxicity, using the mechanism of excessive induction of the membrane signaling of the cell death receptor, accelerate the natural program of cell death by approximately 20% compared to the state of reproductive cells that were not stimulated. Conclusion. At the present stage, one of the tasks of occupational medicine is to study the effect of chemicals on the processes of reproduction, to develop new approaches to assessing the risk of their impact on the reproductive health of workers.

scholarly journals Interplay between Endoplasmic Reticular Stress and Survivin in Colonic Epithelial Cells

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 171 ◽  
Author(s):  
Rohit Gundamaraju ◽  
Ravichandra Vemuri ◽  
Wai Chin Chong ◽  
Stephen Myers ◽  
Shaghayegh Norouzi ◽  
...  
Keyword(s):  
Cell Death ◽  
Cancer Cells ◽  
Inflammatory Markers ◽  
Caspase 3 ◽  
Annexin V ◽  
Parp Cleavage ◽  
Cellular Survival ◽  
Unfolded Protein ◽  
Proliferative Assay ◽  
The Unfolded Protein Response

Sustained endoplasmic reticular stress (ERS) is implicated in aggressive metastasis of cancer cells and increased tumor cell proliferation. Cancer cells activate the unfolded protein response (UPR), which aids in cellular survival and adaptation to harsh conditions. Inhibition of apoptosis, in contrast, is a mechanism adopted by cancer cells with the help of the inhibitor of an apoptosis (IAP) class of proteins such as Survivin to evade cell death and gain a proliferative advantage. In this study, we aimed to reveal the interrelation between ERS and Survivin. We initially verified the expression of Survivin in Winnie (a mouse model of chronic ERS) colon tissues by using immunohistochemistry (IHC) and immunofluorescence (IF) in comparison with wild type Blk6 mice. Additionally, we isolated the goblet cells and determined the expression of Survivin by IF and protein validation. Tunicamycin was utilized at a concentration of 10 µg/mL to induce ERS in the LS174T cell line and the gene expression of the ERS markers was measured. This was followed by determination of inflammatory cytokines. Inhibition of ERS was carried out by 4Phenyl Butyric acid (4PBA) at a concentration of 10 mM to assess whether there was a reciprocation effect. The downstream cell death assays including caspase 3/7, Annexin V, and poly(ADP-ribose) polymerase (PARP) cleavage were evaluated in the presence of ERS and absence of ERS, which was followed by a proliferative assay (EdU click) with and without ERS. Correspondingly, we inhibited Survivin by YM155 at a concentration of 100 nM and observed the succeeding ERS markers and inflammatory markers. We also verified the caspase 3/7 assay. Our results demonstrate that ERS inhibition not only significantly reduced the UPR genes (Grp78, ATF6, PERKandXBP1) along with Survivin but also downregulated the inflammatory markers such as IL8, IL4, and IL6, which suggests a positive correlation between ERS and the inhibition of apoptosis. Furthermore, we provided evidence that ERS inhibition promoted apoptosis in LS174T cells and shortened the proliferation rate. Moreover, Survivin inhibition by YM155 led to a comparable effect as that of ERS inhibition, which includes attenuation of ERS genes and inflammatory markers as well as the promotion of programmed cell death via the caspase 3/7 pathway. Together, our results propose the interrelation between ERS and inhibition of apoptosis assigning a molecular and therapeutic target for cancer treatment.

scholarly journals Analysis of programmed cell death in mouse fetal oocytes

Reproduction ◽  
2007 ◽  
Vol 134 (2) ◽  
pp. 241-252 ◽  
Author(s):  
A M Lobascio ◽  
F G Klinger ◽  
M L Scaldaferri ◽  
D Farini ◽  
M De Felici
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Death Process ◽  
Parp Cleavage ◽  
Tunel Staining ◽  
Calpain Inhibitor ◽  
Dna Ladder ◽  
Caspase 2

We report a short-term culture system that allowsto define novel characteristic of programmed cell death (PCD) in fetal oocytes and to underscore newaspects of this process. Mouse fetal oocytes culturedin conditions allowingmeiotic prophase I progression underwent apoptotic degeneration waves as revealed by TUNEL staining. TEM observations revealed recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features were also observed. Further characterization of oocyte death evidenced DNA ladder, Annexin V binding, PARP cleavage, and usually caspase activation (namely caspase-2). In the aim to modulate the oocyte death process, we found that the addition to the culture medium of the pancaspase inhibitors Z-VAD orcaspase-2-specific inhibitor Z-VDVAD resulted in a partial and transient prevention of this process. Oocyte death was significantly reduced by the antioxidant agent NAC and partly prevented by KL and IGF-I growth factors. Finally, oocyte apoptosis was reduced by calpain inhibitor I and increased by rapamycin after prolonged culture.These results support the notion that fetal oocytes undergo degeneration mostly by apoptosis. This process is, however, often morphologically atypical and encompasses other forms of cell death including caspase-independent apoptosis and autophagia. The observation that oocyte death occurs mainly at certain stages of meiosis and can only be attenuated by typical anti-apoptotic treatments favors the notion that it is controlled at least in part by stage-specific oocyte-autonomous meiotic checkpoints and when activated is little amenable to inhibition being the oocyte able to switch back and forth among different death pathways.

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