The endoplasmic reticulum stress response is involved in apoptosis induced by aloe-emodin in HK-2 cells
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Propidium iodide
MTT assay
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With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8–10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca 2+ (111 ± 14 nM) was elevated in reperfused annexin V-negative cells (214 ± 22 nM), and further elevated in annexin V-positive myocytes (382 ± 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in ∼3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.
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The surface of healthy cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflet of the plasma membrane. One of these lipids, phosphatidylserine (PS), is normally restricted to the inner leaflet of the plasma membrane and is, therefore, only exposed to the cell cytoplasm. However, during apoptosis lipid asymmetry is lost and PS becomes exposed on the outer leaflet of the plasma membrane. Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane. However, apoptotic cells can be distinguished from necrotic cells by co-staining with propidium iodide (PI) because PI enters necrotic cells but is excluded from apoptotic cells. This protocol describes Annexin V binding and PI uptake followed by flow cytometry to detect and quantify apoptotic and necrotic cells.
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Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).
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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.
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Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95% +/- 3.56, p < 0.0001, for 24 hours and mean of 37.67% +/- 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.
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Objective To observe the effect of Spred2 on apoptosis of human hepatocellular carcinoma cells. Methods HepG2 cells were transfected with pcDNA3. 0 and pcDNA3. 0-Spred2 for the morphologic change of HepG2 to be observed. Cells were stained with annexin Ⅴ conjugated to fluorescein isothiocyanate(annexin Ⅴ-FITC) and propidium iodide(PI/7-AAD) . The stained cells were analyzed on a flow cytometer using the Cellquest program. The expression level of Spred2 was examined by Western blot. Results Spred2 over-expression induced HepG2 cells apoptosis and sensitized cells to 5-FU induced HepG2 cells apoptosis. Conclusions Spred2 can regulate HepG2 cellular apoptosis.
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Objective: To investigate the profile of 5-FU-induced apoptosis in tumor cell line and evaluate the usefulness of Annexin V and propidium iodide double staining for detecting apoptosis. Methods: Colon cancer cell line colo320 was incubated with 5-FU of different concentration for 2, 4,6, 8 and 12h respectively, apoptosis was measured using Hoechst33342 staining and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. Results: Apoptosis could be induced in colo 320 cell line by 5-FU,the percentage of AnnexinⅤ~+PI~-cells increased after being treated with 80ug/ml 5-FU for 4 h,both apoptotic and necrotic cell increased at 12h.Apoptotic cells were also identified by Hoechst33342 staining. Conclusion: Apoptosis induced by 5-Fu is time and dose dependent.Annexin V and PI double staining is a specific, sensitive, and quantitative method for analyzing apoptotic cells.
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This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium Iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.
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