scholarly journals Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death

Cytometry ◽  
2004 ◽  
Vol 60A (2) ◽  
pp. 145-154 ◽  
Author(s):  
Gustav Mattiasson
Keyword(s):  
Cell Death ◽  
Flow Cytometric Analysis ◽  
Liver Mitochondria ◽  
Flow Cytometric ◽  
Isolated Liver

Flow Cytometric Analysis of Hyperglycemia-Induced Cell Death in

2021 ◽  
pp. 259-270
Author(s):  
Ravichandra Shivalingappa Davargaon ◽  
M. V. V. Subramanyam ◽  
S. Asha Devi
Keyword(s):  
Cell Death ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric

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 Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells

2016 ◽  
Vol 310 (9) ◽  
pp. L796-L801 ◽  
Author(s):  
Benjamin D. Singer ◽  
Jason R. Mock ◽  
Franco R. D'Alessio ◽  
Neil R. Aggarwal ◽  
Pooja Mandke ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Lung Tissue ◽  
Flow Cytometric Analysis ◽  
Mouse Lung ◽  
Tissue Preparation ◽  
Pulmonary Vascular Permeability ◽  
Flow Cytometric ◽  
Hematopoietic Lineage ◽  
Cellular Compartments

Flow cytometry is a powerful tool capable of simultaneously analyzing multiple parameters on a cell-by-cell basis. Lung tissue preparation for flow cytometry requires creation of a single-cell suspension, which often employs enzymatic and mechanical dissociation techniques. These practices may damage cells and cause cell death that is unrelated to the experimental conditions under study. We tested methods of lung tissue dissociation and sought to minimize cell death in the epithelial, endothelial, and hematopoietic lineage cellular compartments. A protocol that involved flushing the pulmonary circulation and inflating the lung with Dispase, a bacillus-derived neutral metalloprotease, at the time of tissue harvest followed by mincing, digestion in a DNase and collagenase solution, and filtration before staining with fluorescent reagents concurrently maximized viable yields of epithelial, endothelial, and hematopoietic lineage cells compared with a standard method that did not use enzymes at the time of tissue harvest. Flow cytometry identified each population—epithelial (CD326+CD31−CD45−), endothelial (CD326−CD31+CD45−), and hematopoietic lineage (CD326−CD31−CD45+)—and measured cellular viability by 7-aminoactinomycin D (7-AAD) staining. The Dispase method permitted discrimination of epithelial vs. endothelial cell death in a systemic lipopolysaccharide model of increased pulmonary vascular permeability. We conclude that application of a dissociative enzyme solution directly to the cellular compartments of interest at the time of tissue harvest maximized viable cellular yields of those compartments. Investigators could employ this dissociation method to simultaneously harvest epithelial, endothelial, and hematopoietic lineage and other lineage-negative cells for flow-cytometric analysis.

Insights into Nano-Photo-Thermal Therapy of Cancer: The Kinetics of Cell Death and Effect on Cell Cycle

2020 ◽  
Vol 20 (5) ◽  
pp. 612-621
Author(s):  
Mousa Tabei ◽  
Elham Zeinizade ◽  
Jaber Beik ◽  
S. Kamran Kamrava ◽  
Zahra Nasiri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Flow Cytometric Analysis ◽  
Thermal Therapy ◽  
Normal Fibroblast ◽  
Cycle Phase ◽  
Flow Cytometric ◽  
Kinetics Of

Background: Despite considerable advances in nano-photo-thermal therapy (NPTT), there have been a few studies reporting in-depth kinetics of cell death triggered by such a new modality of cancer treatment. Objective: In this study, we aimed to (1) investigate the cell death pathways regulating the apoptotic responses to NPTT; and (2) ascertain the effect of NPTT on cell cycle progression. Methods: Folate conjugated gold nanoparticle (F-AuNP) was firstly synthesized, characterized and then assessed to determine its potentials in targeted NPTT. The experiments were conducted on KB nasopharyngeal cancer cells overexpressing folate receptors (FRs), as the model, and L929 normal fibroblast cells with a low level of FRs, as the control. Cytotoxicity was evaluated by MTT assay and the cell death mode (i.e., necrosis or apoptosis) was determined through AnnexinV/FITC-propidium iodide staining. Next, the gene expression profiles of some key apoptotic factors involved in the mitochondrial signaling pathway were investigated using RT-qPCR. Finally, cell cycle phase distribution was investigated at different time points post NPTT using flow cytometric analysis. Results: The obtained results showed that KB cell death following targeted NPTT was greater than that observed for L929 cells. The majority of KB cell death following NPTT was related to apoptosis. RT-qPCR analysis indicated that the elevated expression of Bax along with the depressed expression of Bcl-xL, Survivin and XIAP may involve in the regulation of apoptosis in response to NPTT. Flow cytometric analysis manifested that 16-24 hours after NPTT, the major proportion of KB cells was in the most radiosensitive phases of the cell cycle (G2/M). Conclusion: This study extended the understanding of the signaling pathway involved in the apoptotic response to NPTT. Moreover, the potential effect of NPTT on sensitizing cancer cells to subsequent radiation therapy was highlighted.

Triggering of CD44 by Ultra-Low-Molecular-Weight Hyaluronan Induces Cell Death by Autophagy Via Endoplasmic Reticulum Stress in Acute Lymphoblastic Leukemia with MLL Gene Rearrangements

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1361-1361 ◽  
Author(s):  
Shin Kasai ◽  
Yoshiyuki Furuichi ◽  
Norie Ando ◽  
Keiko Kagami ◽  
Kumiko Goi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Cell Death ◽  
Er Stress ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Low Molecular Weight ◽  
Gene Rearrangements ◽  
Flow Cytometric ◽  
Induction Of Apoptosis

Abstract Abstract 1361 Acute lymphoblastic leukemia (ALL) with MLL gene rearrangements (MLL+ALL) is chemoresistant and its prognosis is still dismal. MLL+ALL shows a unique gene profile clearly distinguishable from other types of ALL and AML, and CD44 is known as one of specific molecules expressed very highly in MLL+ALL. CD44, a transmembrane glycoprotein recently attracting an attention as a cancer stem cell marker for several types of cancers, is expressed on a variety of cells, and has the specific binding sites for hyaluronic acid (HA, formally hyaluronan). High-molecular-weight (HMW)-HA which exists as extracellular matrix in tissues is incorporated into cells in a CD44-dependent manner, and then is secreted from cells as low-molecular-weight (LMW) to ultra-low-molecular-weight (ULMW)-HA in normal and pathological conditions. The purpose of this study is to investigate the biological events elicited after ligand stimulation of CD44 by using distinct molecular sizes of HA (2.5mg/ml) in MLL+ALL. When cultured in the presence of ULMW-HA (6.4kDa), but not of LMW-HA (31kDa) or HMW-HA (980kDa), thymidine uptakes of cell lines expressing CD44 at very high levels (KOPB-26 and YACL-95) were gradually inhibited in the course of culture from day3 through day5 (greater than 90% decrease at day5), although those expressing CD44 at very low levels (KOCL58, KOCL69) were largely unaffected. Cell cycle analysis of KOPB-26 and YACL-95 using propidium iodide (PI) staining after ULMW-HA stimulation revealed no increase in G0/G1 and G2/M phases and subdiploid population, suggesting that a marked inhibition of thymidine uptakes is neither due to cell cycle arrest nor induction of apoptosis. Dye exclusion test showed a marked decrease in viability at days 3–5 after ULMW-HA stimulation, and flow cytometric analysis showed a gradual increase in the low FSC/high SSC population doubly labeled by Annexin V and PI. However, cytospine smears revealed a massive cell death lacking features characteristic of apoptosis. In addition, pan-caspase inhibitor Z-VAD did not block cell death and cleaved caspase 3 was not detected on flow cytometric analysis after ULMW-HA stimulation, resulting in the conclusion that ULMW-HA-induced cell death is not elicited by induction of apoptosis. Although levels of high-mobility group protein B1 (HMGB1) usually used as a necrosis marker was progressively elevated in culture media after ULMW-HA stimulation, the necrosis inhibitor Necrostatin-1 did not block the ULMW-HA-induced cell death. Finally, electromicroscopic examination was performed and revealed a marked extension of endoplasmic reticulum (ER) and an increase in autophagsomes, and the autophagy inhibitor 3-methyladenine specifically suppressed cell death, indicating that ULMW-HA strongly induces ER stress leading to cell death by autophagy. Changes in ER stress and autophagy-associated molecules after ULMW-HA stimulation were also demonstrated. In conclusion, triggering of CD44 by ULMW-HA stimulation strongly elicits cell death by autophagy via ER stress in chemoresistant MLL+ALL. CD44-targeted therapy by ULMW-HA possibly becomes a useful strategy not only for MLL+ALL but also for CD44-high hematological and non-hematological tumors particularly for those in which the cancer stem cell population is highly positive for CD44. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Triazole Hybrid of Neolignans as a Potential Antileishmanial Agent by Triggering Mitochondrial Dysfunction

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 37 ◽  
Author(s):  
Carla Cardozo Pinto de Arruda ◽  
Daiana de Jesus Hardoim ◽  
Yasmin Silva Rizk ◽  
Celeste da Silva Freitas de Souza ◽  
Tânia Zaverucha do Valle ◽  
...  
Keyword(s):  
Cell Death ◽  
Direct Action ◽  
Flow Cytometric Analysis ◽  
Tem Analysis ◽  
Methylenedioxy Group ◽  
Highly Active ◽  
Flow Cytometric ◽  
Transmission Electron ◽  
New Compounds ◽  
Intracellular Amastigote

In the search for new compounds with antileishmanial activity, we synthesized a triazole hybrid analogue of the neolignans grandisin and machilin G (LASQUIM 25), which was previously found highly active against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. In this work, we investigated the leishmanicidal effects of LASQUIM 25 to identify the mechanisms involved in the cell death of L. amazonensis promastigotes. Transmission electron microscopy (TEM) analysis showed marked effects of LASQUIM 25 (IC50 = 7.2 µM) on the morphology of promastigote forms, notably on mitochondria. The direct action of the triazole derivative on the parasite was noticed over time from 2 h to 48 h, and cells displayed several ultrastructural alterations characteristic of apoptotic cells. Also, flow cytometric analysis (FACS) after TMRE staining detected changes in mitochondrial membrane potential after LASQUIM 25 treatment (64.83% labeling versus 83.38% labeling in nontreated cells). On the other hand, FACS after PI staining in 24 h-treatment showed a slight alteration in the integrity of the cell membrane, a necrotic event (16.76% necrotic cells versus 3.19% staining in live parasites). An abnormal secretion of lipids was observed, suggesting an exocytic activity. Another striking finding was the presence of autophagy-related lysosome-like vacuoles, suggesting an autophagic cell death that may arise as consequence of mitochondrial stress. Taken together, these results suggest that LASQUIM 25 leishmanicidal mechanisms involve some degree of mitochondrial dysregulation, already evidenced by the treatment with the IC50 of this compound. This effect may be due to the presence of a methylenedioxy group originated from machilin G, whose toxicity has been associated with the capacity to generate electrophilic intermediates.

scholarly journals Flow Cytometry Reveals the Nature of Oncotic Cells

2019 ◽  
Vol 20 (18) ◽  
pp. 4379
Author(s):  
Anna Vossenkamper ◽  
Gary Warnes
Keyword(s):  
Cell Death ◽  
Sodium Azide ◽  
Caspase 3 ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Regulated Cell Death ◽  
Damage Response ◽  
Induced Apoptosis ◽  
Apoptosis And Necrosis ◽  
Specific Pathway

The term necrosis is commonly applied to cells that have died via a non-specific pathway or mechanism but strictly is the description of the degradation processes involved once the plasma membrane of the cell has lost integrity. The signalling pathways potentially involved in accidental cell death (ACD) or oncosis are under-studied. In this study, the flow cytometric analysis of the intracellular antigens involved in regulated cell death (RCD) revealed the phenotypic nature of cells undergoing oncosis or necrosis. Sodium azide induced oncosis but also classic apoptosis, which was blocked by zVAD (z-Vla-Ala-Asp(OMe)-fluoromethylketone). Oncotic cells were found to be viability+ve/caspase-3–ve/RIP3+ve/–ve (Receptor-interacting serine/threonine protein kinase 3). These two cell populations also displayed a DNA damage response (DDR) phenotype pH2AX+ve/PARP–ve, cleaved PARP induced caspase independent apoptosis H2AX–ve/PARP+ve and hyper-activation or parthanatos H2AX+ve/PARP+ve. Oncotic cells with phenotype cell viability+ve/RIP3–ve/caspase-3–ve showed increased DDR and parthanatos. Necrostatin-1 down-regulated DDR in oncotic cells and increased sodium azide induced apoptosis. This flow cytometric approach to cell death research highlights the link between ACD and the RCD processes of programmed apoptosis and necrosis.

Sign in / Sign up

Export Citation Format

Share Document