Study on Comparison of Methods for Detection of Cell Apoptosis by Flow Cytometry
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To evaluate the method of measuring apoptosis by flow cytometry with three stains, the apoptosis percentage of rat cortical neurons was measured by flow cytometry using propidium iodide(PI) staining, Annexin V/PI and JC-1 double staining respectively. The results showed that: the apoptotic positive percentage measured by PI staining,Annexin V/PI and JC-1 was 2.60%,6.52% and 18.56%, respectively. Our conclusion is that there were significant differences among the results of three staining methods: JC-1 staining was the most sensitive method, whereas PI single staining was not suitable to measure the early apoptosis of cells.Keywords:
Propidium iodide
Cytometry
Annexin A5
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Cytometry
Negative stain
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Objective To investigate the factors that may influence the position of hypodiploid peak in the histogram describing cell apoptosis using flow cytometry with PI staining. Methods Cellular apoptosis was induced by dexamethasone in the thymus lymphocytes of SD rats, which were fixed in different fixatives (70% ethanol, 4% formaldehyde, 4% paraformaldehyde, 1% paraformaldehyde and 70% ethanol in sequence) for 1 to 24 h at different temperatures (4℃, -20℃and 37℃) and suspended in suspension buffer for 1 to 24 h. The cells subjected to the above treatments were studied using flow cytometry with PI staining. Results The hypodiploid peak appeared in the histogram only when apoptotic thymus lymphocytes were fixed by 70% ethanol alone. The temperature for fixation and time period of suspension had an obvious influence on the position of hypodiploid peak, but time period of fixation had little effect. Conclusion The detection of cell apoptosis by flow cytometry with PI staining is influenced by fixatives, time period and temperature for fixation and time period of suspension. PI staining, however, is a simple, perceptible method for quantitatively detecting apoptotic cells in culture so long as the conditions are appropriately controlled.
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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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Objective To investigate the property of apoptosis of HepG2 cell line induced by CDDP.Methods Liver cancer cell line HepG2 was incubated with CDDP of different concentrations for 4 h,15 h,24 h,36 h,respectively.Apoptosis was measured by flow cytometry method and Annexin V and PI double staining.Results Apoptosis of HepG2 cell could be induced by CDDP,the percentage of AnnexinⅤ+ PI cells increased along with the increase of CDDP concentration and incubation time.Conclusions Apoptosis of HepG2 cell induced by CDDP was time and dose dependent.Annexin V and PI double staining could be a specific method for analyzing apoptotic cells.
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Objective To evaluate Annexin V technique for measuring hepatic apoptosis and to investigate the protective function of Rg1 and Rb1 on acute liver injury. Methods LPS-treated acute liver injury and the protective effect of Rg1 and Rb1 were assessed by Annexin V double staining and PI staining measurements. The activity of sPLA2 was measured by [3H]-labelled oleic acid method. Results Annexin V showed a higher sensitivity and specificity than PI staining. It was only the Annexin V assay that could discriminate normal cells, apoptosis cells, and necrotic cells. Rg1 and Rbl could reduce the percentage of hepatic apoptotic and necrotic cells (P0.01) and activity of sPLA2 (p0.01). Conclusions The Annexin V assay is an ideal method for measuring apoptosis presently. Rgl and Rbl have a definite protective effect on acute liver injury in rats.
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Propidium iodide
Terminal deoxynucleotidyl transferase
DNA laddering
Jurkat cells
Fluorescein isothiocyanate
Rhodamine 123
Annexin A5
Apoptotic DNA fragmentation
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Objective.To quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions. Methods.The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1.0 μ mol/L dexamethasone (DEX) for 2,4 and 8 h respectively,then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detect the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis,and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosis,then apoptotic cells were quantified by flow cytometry (FCM).Furthermore,Annexin+ /PI and Annexin+ /PI+ cells were sorted by fluoresence activated cell sorter (FACS),and identified by electron microscopy (EM) and DNA gel electrophoresis. Results.The percentage of apoptotic cells was found to increase with the incubation time (r=0.97).This method was sensitive with low detection limit (0.02% ),and was reproducible with low coefficient variance (CV)(4.2% ).Meanwhile,the Annexin+ /PI and Annexin+ /PI+ cells were identified as apoptotic and necrotic cells under EM,and DNA extracted from the Annexin+ /PI cells was characterized by ladder pattern . Conclusions.Annexin V assay is a specific,sensitive,accurate,reproductive and quantitative method for analyzing apoptotic cells.
Propidium iodide
Annexin A5
Fluorescein isothiocyanate
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To evaluate the merits and demerits of annexin V-FITC/PI and Hoechst33342/PI double stainings in the detection of apoptosis by flow cytometry.Hoechst33342/PI and annexin V-FITC/PI double stainings were performed to detect the apoptosis of thymocytes induced by DEX and K562 cells induced by H(2)O(2).The anenxin V-FITC/PI double staining was able to detect the cell apoptosis in both DEX-induced thymocytes and H(2)O(2)-induced K562 cells as predicted, and the cell plotting was consistent with the principle of the method. Hoechest33342/PI double staining was able to detect cell apoptosis in the DEX-induced thymocytes, but this method showed a contradictory result in the early stage of H(2)O(2)-induced K562 cell apoptosis, and the cell plotting was in conflict with the principle of the method.Compared with annexin V-FITC/PI double staining, the method of Hoechst33342/PI double staining in the detection of apoptosis by flow cytometry has some limitations.
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Background We have previously characterized apoptotic cell death induced in a follicular lymphoma cell line, HF-1, after triggering via the B-cell receptor (BCR) or treatment with Ca2+ Ionophore A23187. We analyzed the kinetics of apoptosis induced by these two treatments, as two alternative models of classical apoptosis, by flow cytometry using a novel combination of cytofluorometric stains. Methods Cells were stained with a combination of Annexin V-FITC, propidium iodide (PI), and SYTO 17 and analyzed by a two-laser flow cytometry system using 488-nm argon and 633-nm HeNe air-cooled lasers. Results In both apoptotic models, the first apoptotic cells were detected by SYTO 17 staining. The alteration in SYTO 17 staining intensity was followed by an increased uptake of PI. Finally, the apoptotic cells were labeled with Annexin V in BCR-induced apoptosis. On the contrary, on treatment with Ca2+ Ionophore A23187, cells became positive for Annexin V earlier than for PI. Conclusions The novel cytofluorometric dye, SYTO 17, discriminates apoptotic alterations before Annexin V and PI. PI also discriminates apoptotic alterations before the loss of plasma membrane asymmetry by BCR but not by Ca2+ Ionophore A23187-induced apoptosis. Finally, the combination of these three cytofluorometric dyes allows effective detection of apoptotic subpopulations and ordering of apoptotic events by flow cytometry. Cytometry 43:134–142, 2001. © 2001 Wiley-Liss, Inc.
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Cytometry
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