Flow cytometric evaluation of apoptosis and cell viability as a criterion of anti-tumour drug toxicity.
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Determination of the drug concentration required to kill 50% of the tumour cells (EC50) does not take into account the propensity of cells to undergo apoptosis and necrosis. These 2 parameters and the viable cells are here assessed by a flow cytometric (FC) approach using propidium iodide (PI) and FITC-Annexin V staining.A number of carcinoma cell lines of defined p53 status were exposed to cis-PtII for 24 hours, stained with PI and FITC-Annexin V and analyzed by FC. Unstained viable cells, early apoptotic cells and necrotic cells were scored separately in dual parameter plots of green fluorescence (FITC) against red fluorescence (PI) to generate dose response curves.EC50 values for cell viability were found to be 1-4 times higher than survival data from colony assays resembling data obtained by MTT or Crystal Violet vital dye staining. Percentage apoptosis measured by Annexin V binding was in agreement with microscopic scoring of apoptotic cells after Acridine Orange staining.The FC assay described gives a good estimate of cell viability resembling data from vital dye staining assays and provides additional information on apoptosis and necrosis. FC data from Annexin V binding and microscopic scoring after Acridine Orange staining were in excellent agreement.Keywords:
Acridine orange
Propidium iodide
Viability assay
Annexin A5
MTT assay
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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.
Propidium iodide
Acridine orange
Trypan blue
Ethidium bromide
Giemsa stain
Annexin A5
DAPI
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Determination of the drug concentration required to kill 50% of the tumour cells (EC50) does not take into account the propensity of cells to undergo apoptosis and necrosis. These 2 parameters and the viable cells are here assessed by a flow cytometric (FC) approach using propidium iodide (PI) and FITC-Annexin V staining.A number of carcinoma cell lines of defined p53 status were exposed to cis-PtII for 24 hours, stained with PI and FITC-Annexin V and analyzed by FC. Unstained viable cells, early apoptotic cells and necrotic cells were scored separately in dual parameter plots of green fluorescence (FITC) against red fluorescence (PI) to generate dose response curves.EC50 values for cell viability were found to be 1-4 times higher than survival data from colony assays resembling data obtained by MTT or Crystal Violet vital dye staining. Percentage apoptosis measured by Annexin V binding was in agreement with microscopic scoring of apoptotic cells after Acridine Orange staining.The FC assay described gives a good estimate of cell viability resembling data from vital dye staining assays and provides additional information on apoptosis and necrosis. FC data from Annexin V binding and microscopic scoring after Acridine Orange staining were in excellent agreement.
Acridine orange
Propidium iodide
Viability assay
Annexin A5
MTT assay
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The growth inhibition and pro-apoptosis effects of dracorhodin perchlorate on human prostate cancer PC-3 cell line were examined. After administration of 10-80 μmol/L dracorhodin perchlorate for 12-48 h, cell viability of PC-3 cells was measured by MTT colorimetry. Cell proliferation ability was detected by colony formation assay. Cellular apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent staining, and flow cytometry (FCM) with annexin Ⅴ-FITC/propidium iodide dual staining. The results showed that dracorhodin perchlorate inhibited the growth of PC-3 in a dose- and time-dependent manner. IC50 of dracorhodin perchlorate on PC-3 cells at 24 h was 40.18 μmol/L. Cell clone formation rate was decreased by 86% after treatment with 20 μmol/L of dracorhodin perchlorate. Some cells presented the characteristic apoptotic changes. The cellular apoptotic rates induced by 10-40 μmol/L dracorhodin perchlorate for 24 h were 8.43% to 47.71% respectively. It was concluded that dracorhodin perchlorate significantly inhibited the growth of PC-3 cells by suppressing proliferation and inducing apoptosis of the cells.
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Acridine orange
Perchlorate
Ethidium bromide
MTT assay
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Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.
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Acridine orange
Ethidium bromide
Trypan blue
Annexin A5
Agarose gel electrophoresis
Viability assay
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Abstract Background: The aim of this study was to investigate by flow cytometry cellular viability and apoptosis of human chondrocytes isolated from osteoarthritic cartilage and to correlate replicative senescence with apoptosis of these cells. Methods: To understand the mechanisms underlying the process of cell death in cartilage destruction, we investigated by flow cytometry cellular viability (Cell viability calcein‐AM assay) and apoptosis (Light scattering properties of chondrocytes, study of chondrocyte death using Annexin‐V‐FITC and propidium iodide double‐labeling, caspase‐3 activity determination) of human chondrocytes isolated from osteoarthritic and nonosteoarthritic cartilage. Senescent cells were characterized using the senescence‐associated‐β‐galactosidase marker (SA‐β‐Gal marker) by staining with chromogenic substrate (X‐Gal) to produce blue coloration of SA‐β‐Gal‐positive cells and microscopy analysis. Results: The results we obtained show that between 25 and 40% of chondrocytes were in apoptosis and all of them were SA‐β‐Gal‐positive. Conclusions: These results demonstrate that the death of osteoarthritic chondrocytes is an apoptotic phenomenon which is preceded by an accelerated mechanism of replicative senescence. © 2008 Clinical Cytometry Society
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Senescence
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Objective To study the apoptosis-inducing effect of gambogenic acid(GNA)combined with adriamycin(ADM)on human breast cancer MCF-7cells.Methods Human breast cancer MCF-7cells were cultured invitro.Cell viability was determined by MTT assay;cell morphology was observed by inverted microscopy;nuclear staining with 4′,6-diamidino-2-phenylindole was used to assess the effect of GNA and ADM used alone or together on the apoptosis of MCF-7cells;annexin V(AV)-fluorescein isothiocyanate(FITC)/propidium iodide(PI)staining and flow cytometry were used to measure the apoptosis rate of MCF-7cells treated with GNA and/or ADM.Results The MTT assay showed that the viability of MCF-7cells decreased as the concentrations of GNA and ADM increased within 0.125-4μmol/L and0.5-16μmol/L,respectively,and the cell viability was lower when GNA and ADM were used together than when they were used alone.The inverted microscopy and fluorescence microscopy showed that,compared with control cells,treated cells became markedly round and small,had broken nuclei,and were apoptotic,and the number of apoptotic cells was larger when both GNA and ADM were used than when they were used alone.AV-FITC/PI staining and low cytometry showed that GNA and/or ADM induced the apoptosis of MCF-7cells,and the apoptosis rate increased significantly when GNA and ADM were used together than when they were used alone(P0.01).Conclusion The single and combined use of GNA and ADM can induce the apoptosis of human breast cancer MCF-7cells,and the combination of two drugs has a synergistic effect.The mechanism may be related to inducing cell apoptosis.
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Viability assay
MCF-7
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Annexin A5
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Treatment of diseases with synthetic materials has been an aspiration of mankind since the dawn of human development. In this research, three complex compounds of azamacrocycle (TD1, TD2, and TD3) were synthesized, and experiments were conducted to determine whether their toxicity to human liver carcinoma (HepG2 ) cells is associated with apoptotic and/or necrotic cell death. Cell survival was determined by MTT assay. Apoptosis and necrosis were measured by annexin V FITC/PI assay using the flow cytometry and by propidium iodide (PI) assay using the cellometer vision. HepG2 cells were treated with different concentrations of azamacrocycles for 48 h. Results from MTT assay indicated that all the three azamacrocycles significantly (p < 0.05) reduce cell viability in a dose-dependent manner, showing 48 h-LD50 values of about 37.97, 33.60, and 19.29 μM, for TD3, TD1 and TD2, respectively. Among the three compounds tested, TD2 showed the most pronounced cytotoxic activity against HepG2 cells, being about twofold more potent than TD3. The order of toxicity was TD2 > TD1 > TD3. Because TD2 exerted the most cytotoxic activity against HepG2 cells, it was used in the subsequent apoptosis and necrosis-related experiments. The flow cytometry assessment showed a strong dose-response relationship with regard to TD2 exposure and annexin V/PI positive cells. PI assay data indicated that TD2 exposure increased the proportion of fluorescence positive cells. Overall, our results indicate that azamacrocycle toxicity to HepG2 cells is associated with apoptotic and necrotic cell death resulting from phosphatidylserine externalization and loss of membrane integrity.
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Flow cytometry was used to quantify the percentage of apoptotic hepatic Caracimoma cells infected with bluetongue virus HbC3 and stained with propidium iodide (PI) and Annexin V/PI. The percentages of the apoptotic cells measured by PI at 24h, 36h and 48h were 10.8 ±3.05, 21.7±6.28, 28.3±10.6, respectively, and those by Annexin V-PI were 20.42±3.70, 49.3±8.11, 79.6±11.5, respectively. The two staining methods and the rate of apoptotic cells infected by bluetengue virus hours were significantly different (P0.01). The early apoptotic cells and secondary necrosis could be detected by AnnexinV/PI assay with good sensitivity and specificity. The apoptotic cells induced by bluetengue virus was one of the important forms in the pathological changes and cell necrosis.
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Background: Inflammation is a complex physiopathologic response to different stimuli. Recently, some pharmacological strategies have been proposed that could be used for resolution of inflammation by enhancing apoptosis of inflammatory cells. Objectives: To study in vitro apoptotic activity of isoespintanol [ISO] and of two semi-synthetic derivatives, bromide isoespintanol [BrI] and demethylated isoespintanol [DMI], in human polymorphonuclear (PMN) cells. Methods: PMN were exposed to the different concentrations of ISO, BrI and DMI for 30 min in phosphate-buffered saline pH 7.4 containing 1 mg/mL glucose, 0.4 mM Mg2+, and 1.20 mM Ca2+. Viability was assessed by dimethylthiazol diphenyl tetrazolium bromide (MTT). To distinguish between the two modes of cell death, apoptosis and necrosis, we examined differences in morphological and biochemical changes of cells stained with annexin V- FITC (An) and/or propidium iodide (PI) using two different assays based on flow cytometry Results: The MTT assay revealed the ability of cells to reduce MTT salt to formazan. In the presence of BrI and DMI a significant concentration-dependent decrease of cell viability was observed. The annexin V- FITC binding assay showed a high proportion of apoptotic cells for those treated with BrI (An+/ PI-: 62.3 ± 8.2% vs. 2.1 ± 0.5% of control, P<0.05). The population of PMN treated with DMI produced the highest percentage (An+/IP+: 43.4 ± 5.2 % vs. 0.4 ± 0.3 % of control, P<0.05) of necrotic cells. Apoptotic nuclei were analyzed by PI staining. The cell population in the sub G0/G1 region represents cells with hypodiploidal DNA, an indicator of apoptosis. When cells were incubated with 50 and 100 μM of BrI, the cell population in the sub G0/G1 region increased, suggesting a dose-dependent increase in the population of apoptotic cells. The presence of the pan-inhibitor of caspases (Z-VAD-fmk) showed a significant reduction in cell population in the sub G0/G1 region, indicating less degradation of DNA. Conclusions: Bromide isoespintanol [BrI] induces an apoptotic process in PMN, mediated –at least in part– by activation of caspases, although this compound may probably act through other caspase-independent mechanisms as well.
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Formazan
MTT assay
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