Evaluation of apoptosis and necrosis induced by statins using fluorescence-enhanced flow cytometry

Background: The search for novel metallic chemical compounds with toxicogenic effects have been of great importance for more efficient cancer treatment. Objective: The study evaluated the cytotoxic, genotoxic and mutagenic activity of organoteluran RF07 in S-180 cell line. Methods: The bioassays used were cell viability with 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, evaluation of apoptosis and necrosis using fluorescence and flow cytometry, cytokinesis-block micronucleus test and comet assay. The compound was tested at 1; 2.5 and 5 µM. Results: The results showed the cytotoxicity of RF07 at concentrations of 2.5, 5, 10 and 20 µM when compared to the negative control. For genotoxicity tests, RF07 showed effects in all concentrations assessed by increased index and frequencies of damage and mutagenic alterations. The compound was also cytotoxic due to the significant decrease in nuclear division index, with significant values of apoptosis and necrosis. The results of fluorescence and flow cytometry showed apoptosis as the main type of cell death caused by RF07 at 5 µM, which is thought to avoid an aggressive immune response of the organism. Conclusion: In addition to cytotoxic and genotoxic effects, RF07 creates good perspectives for future antitumor formulations.

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

Analytical Cellular Pathology ◽

10.1155/1999/176515 ◽

1999 ◽

Vol 19 (3-4) ◽

pp. 139-151 ◽

Cited By ~ 108

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.

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Quantification of Apoptosis and Necrosis by Flow Cytometry

Acta Oncologica ◽

10.3109/02841869309093620 ◽

1993 ◽

Vol 32 (4) ◽

pp. 417-424 ◽

Cited By ~ 52

Author(s):

Michael G. Ormerod ◽

Xiao-Ming Sun ◽

David Brown ◽

Roger T. Snowden ◽

Gerald M. Cohen

Keyword(s):

Flow Cytometry ◽

Apoptosis And Necrosis

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Cell apoptosis and necrosis studied by flow cytometry

Biology of the Cell ◽

10.1016/0248-4900(92)90325-u ◽

1992 ◽

Vol 76 (2) ◽

pp. 247-247

Author(s):

Zbigniew Darzynkiewicz

Keyword(s):

Flow Cytometry ◽

Cell Apoptosis ◽

Apoptosis And Necrosis

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

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot087288 ◽

2016 ◽

Vol 2016 (11) ◽

pp. pdb.prot087288 ◽

Cited By ~ 56

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

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Anticancer Activity of Methanol Extract of Ficus carica Leaves and Fruits Against Proliferation, Apoptosis, and Necrosis in Huh7it Cells

Cancer Informatics ◽

10.1177/1176935119842576 ◽

2019 ◽

Vol 18 ◽

pp. 117693511984257 ◽

Cited By ~ 6

Author(s):

Risa Purnamasari ◽

Dwi Winarni ◽

Adita Ayu Permanasari ◽

Eva Agustina ◽

Suhailah Hayaza ◽

...

Keyword(s):

Flow Cytometry ◽

Anticancer Activity ◽

Anticancer Agents ◽

Antioxidant Activities ◽

Ficus Carica ◽

Anticancer Activities ◽

Leaf Extracts ◽

Fruit Extracts ◽

The Difference ◽

Apoptosis And Necrosis

The polyphenol plant extracts have previously been demonstrated to act as chemopreventive and anticancer agents. Ficus carica is a rich source of polyphenols, yet its antioxidant and anticancer activities remain poorly characterized. This study aimed to determine the anticancer activity of F carica leaf and fruit extracts by investigating their impact on proliferation, apoptosis, and Huh7it cell necrosis. Leaves and fruits were extracted using methanol, and the phytochemical contents were analyzed using Fourier-transform infrared spectroscopy. The antioxidant activity was measured using the 2,2-diphenyl-1-picrylhydrazyl method. Anticancer activities were examined through MTT (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay on Huh7it liver cancer cells. The apoptosis and necrosis conditions were examined using Annexin biomarkers V-PI and later analyzed in flow cytometry. F carica leaves and fruit examined were found to have strong antioxidant activities with IC50 values of 7.9875 µg/mL and 13.402 µg/mL, respectively. MTT assay results indicated F carica leaves and fruit had IC50 values >653 μg/mL and >2000 μg/mL, respectively. The flow cytometry analysis indicated a higher percentage of Huh7it apoptosis and necrosis in leaf extracts compared with fruit extracts. The difference in anticancer activity was attributed to differing compounds present in each extract.

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Dissociation of chicken blastoderm for examination of apoptosis and necrosis by flow cytometry

Poultry Science ◽

10.3382/ps.2009-00552 ◽

2010 ◽

Vol 89 (5) ◽

pp. 901-909 ◽

Cited By ~ 21

Author(s):

J.A. Hamidu ◽

A.M. Rieger ◽

G.M. Fasenko ◽

D.R. Barreda

Keyword(s):

Flow Cytometry ◽

Apoptosis And Necrosis

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Effect of Isolation Techniques on Viability of Bovine Blood Neutrophils

Acta Veterinaria Brno ◽

10.2754/avb200675030343 ◽

2006 ◽

Vol 75 (3) ◽

pp. 343-353 ◽

Cited By ~ 5

Author(s):

P. Sláma ◽

Z. Sládek ◽

D. Ryšánek

Keyword(s):

Flow Cytometry ◽

Light Microscopy ◽

Neutrophil Granulocytes ◽

Bovine Blood ◽

Isolation Techniques ◽

Erythrocyte Lysis ◽

Isolation Methods ◽

Blood Neutrophils ◽

Hypotonic Lysis ◽

Apoptosis And Necrosis

The effect of selected isolation methods on the viability of neutrophil granulocytes (neutrophils) from the blood of healthy Holstein x Bohemian Red Pied crossbred heifers was evaluated. Two methods of neutrophil isolation were used: a) neutrophil isolation on the basis of hypotonic erythrocyte lysis (in two variants: after the erythrocyte lysis proper, the cells were centrifuged at either 200 g or 1000 g), and b) neutrophil isolation with FACS Lysing Solution as the lysing agent. The viability of the isolated neutrophils was evaluated on the basis of apoptosis and necrosis. The results obtained with flow cytometry (FCM) suggest that, from the isolation techniques used, the method based on FACS Lysing Solution impaired the neutrophil viability least. After the application of this method, 5.36 ± 2.15% of neutrophils were apoptotic and 0.51 ± 0.12% were necrotic. In contrast, when the hypotonic erythrocyte lysis was used, the proportion of apoptotic neutrophils amounted to 42.14 ± 7.12% and 49.00 ± 14.70%, respectively, and 41.12 ± 5.55% and 36.91 ± 24.38% respectively of necrotic neutrophils (P < 0.01). This was also confirmed by the light microscopy. After the isolation with FASC Lysing Solution, 1.92 ± 1.74% of neutrophils were apoptotic and 1.05 ± 0.76% were necrotic, as distinct from after the hypotonic erythrocyte lysis where 9.43 ± 3.69% of neutrophils were apoptotic and 12.67 ± 4.74% of necrotic after centrifugation at 200 g, while 12.60 ± 4.35 were apoptotic and 14.96 ± 12.64% were necrotic after centrifugation at 1000 g. It follows from the above-mentioned data that hypotonic lysis is not a suitable method for the isolation of neutrophils, as the method itself markedly affects cell viability.

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Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis

Thrombosis and Haemostasis ◽

10.1160/th05-07-0463 ◽

2005 ◽

Vol 94 (12) ◽

pp. 1236-1244 ◽

Cited By ~ 11

Author(s):

Olav Klingenberg ◽

Reidun Øvstebø ◽

Gun-Britt Joø ◽

Åse-Brit Westvik ◽

Peter Kierulf ◽

...

Keyword(s):

Flow Cytometry ◽

Tissue Factor ◽

Procoagulant Activity ◽

Clinical Samples ◽

Meningococcal Infection ◽

High Concentrations ◽

Apoptosis And Necrosis ◽

Dose Dependent Increase ◽

Dose Dependent

SummaryTissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.

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Preconditioning attenuates apoptosis and necrosis: role of protein kinase Cε and -δ isoforms

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.1.h404 ◽

2001 ◽

Vol 281 (1) ◽

pp. H404-H410 ◽

Cited By ~ 55

Author(s):

Huiping Liu ◽

Bradley C. McPherson ◽

Zhenhai Yao

Keyword(s):

Flow Cytometry ◽

Protein Kinase ◽

Simulated Ischemia ◽

Kinase C ◽

Pkc Inhibitors ◽

Complementary Techniques ◽

Apoptosis And Necrosis

Preconditioning reduces cardiomyocyte necrosis in vivo and in vitro, but it is unknown whether preconditioning blocks apoptosis. We wanted to compare the effects of preconditioning on necrosis and apoptosis in cardiomyocytes. Necrosis was detected with propidium iodide, and apoptosis was quantified by three complementary techniques: flow cytometry, TdT-mediated dUTP nick-end labeling assay, and DNA-laddering electrophoresis. Apoptosis increased with simulated ischemia time (6 h, 19 ± 1%; 12 h, 27 ± 2%; 18 h, 40 ± 4%; 24 h, 54 ± 4%; and 36 h, 83 ± 4%; n = 6 for each group). Simulated ischemia and reoxygenation contributed equally to apoptosis (12-h ischemia, 27 ± 2%, n = 6; 12-h ischemia and 12-h reoxygenation, 51 ± 4%, n = 6; and 24-h ischemia, 54 ± 5%, n = 8). Necrosis occurred primarily during reoxygenation; none was detected during simulated ischemia. Preconditioning with 10 min of simulated ischemia reduced necrosis (18 ± 6%, n = 8) but had no effect on apoptosis. However, three 1-min cycles of simulated ischemia separated by 5 min of reoxygenation reduced necrosis and apoptosis similarly. The protein kinase C (PKC) inhibitors Go6976 (0.1 μM) or chelerythrene (4 μM) abolished the effect of preconditioning. Preconditioning selectively activated PKCε but had no effect on PKCδ and on total PKC enzyme activity. Preconditioning protected against necrosis and apoptosis, but the preconditioning ischemia required for blocking apoptosis was less than that for reducing necrosis. Activation of PKCε isoform is important in mediating the protection.

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