Morphologic assessment of apoptotic cells was performed using the AO/EB staining method. NCI-H295R and SW-13 cells were cultured in 6-well plates (5 × 104 cells/ml). After a 24-h incubation, the cells were treated with rottlerin in the dark (concentration = 5 μM and 2.5 μM) for 48 h. After washing twice with PBS, the cells were stained with 1ml of AO/EB (Sigma Aldrich) for 3min and imaged under an inverted fluorescence microscope. Five hundred cells were counted under a microscope (magnification = 200×). The apoptosis rate was calculated using the following formula: apoptosis rate = (early apoptotic cells + late apoptotic cells)/the total number of cells. The nuclei of early apoptotic cells had a bright green fluorescence, the late apoptotic cell nuclei had a bright orange fluorescence, and the nuclei of normal cells had a ground glass-like dim green fluorescence.
Ao eb
Manufactured by Merck Group
Sourced in United States, Japan
The AO/EB is a laboratory instrument used for the analysis of cell viability and cytotoxicity. It utilizes a combination of acridine orange (AO) and ethidium bromide (EB) dyes to stain cells, allowing for the differentiation between live and dead cells through fluorescent microscopy.
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Lab products found in correlation
Eclipse ti, by Nikon (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Fv300 fv500 laser scanning confocal microscope, by Olympus (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Hemocytometer, by Revvity (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescin diacetate dcfda, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Beclin 1, by Santa Cruz Biotechnology (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
39 protocols using ao eb
1
Apoptosis Evaluation via AO/EB Staining
2
Apoptosis Assessment in Breast Cancer Cells
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MCF-7 and ZR-75-1 breast cancer cells were grown and treated as previously described for the trypan blue dye exclusion assay. At the end of treatment, cells were harvested with the help of trypsin and the cell suspension (25 µL = 500 cells), which was transferred to a glass slide. Then, dual fluorescent staining solution (1 µL) containing 50 µg/mL AO (Acridine orange) and 50 µg/mL EB (Ethidium bromide) (AO/EB, Sigma, St. Louis, MO) was added to each suspension and covered with a coverslip. The morphology of the apoptotic cells was examined, and 500 cells were counted within 20 min using a fluorescent microscope (Olympus Medical Systems India Private Limited, Gurgaon, India).
3
Osteosarcoma Cell Apoptosis Assay
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After obtaining IRB approval, a human osteosarcoma cell line (OS-732 cells, purchased from Beijing JiShuiTan Hospital, Beijing, China) in the logarithmic growth phase were digested with 0.25% trypsin (Hyclone, Logan, UT). RPMI1604 culture medium (Hyclone, Logan, UT) containing 10% fetal calf serum (FCS, Hyclone, Logan, UT) was deposited in each well of a 96-well plate (100 μl/well). Cells were added to a final concentration of 2×104/ml, and the plates were incubated. Cells were left untreated or treated with 30, 60, or 120 μg/ml of kappa-selenocarrageenan (Shanghai Tiancifu Biological engineering Co. Ltd., Shanghai, China). The samples in a 96-well plate were divided into 4 groups, with 24 well samples in each group corresponding to different reagent concentrations. After being cultured for 24 h, 48 h, and 4 d, 20 μl of trypsin was added into each well. When cells had sloughed off, suspensions (25 μl) were transferred to glass slides. Dual fluorescent staining solution (1 μl) containing 100 μg/ml AO and 100 μg/ml EB (AO/EB, Sigma, St. Louis, MO) was added to each suspension and then covered with a coverslip. The morphology of apoptotic cells was examined and 500 cells were counted within 20 min using a fluorescent microscope (OLYMPUS, Japan). Dual acridine orange/ethidium bromide (AO/EB) staining method was repeated 3 times at least.
4
AO/EB Staining for Cell Viability
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Viability of the IECs treated with or without H2O2 was evaluated by double staining with AO/EB (Sigma). After removing the H2O2, the cells were washed with PBS and trypsinized. A mixture containing 100 µL of the cell suspension containing about 105 cells/mL in PBS and 10 µL of the AO/EB working solution (50 µg/mL of AO and 50 µg/mL of EB in PBS) was then prepared. The cells were immediately analyzed with a fluorescence microscope (Olympus IX71; Olympus Corporation). Cell death rate was monitored by trypan blue exclusion with a hemocytometer (Nexcelom Bioscience, USA) as previously described [19 (link)].
5
Quantifying Apoptosis in Cancer Cells
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Normal, early apoptotic, late apoptotic and necrotic cells may be detected by fluorescence microscopy with green fluorescence. The HCT-116 cells were seeded at a density of 5×105 cells/well in 6-well plates in which a round cover slide was placed, followed by a 24-h incubation at 37°C. Subsequently, the cells were treated with different concentrations of curcumin and 5-FU for 24 h. The cover slides to which the cells attached were stained with 20 µl AO/EB (Sigma-Aldrich; Merck KGaA) and observed under a green fluorescence microscope (Nikon 80i; Nikon Corporation) at a magnification of ×200. A total of 1,000 cells in each group were counted, and the apoptosis rate was calculated according to the following equation: Apoptosis rate=(early apoptotic cells + late apoptotic cells)/1,000 ×100%.
6
PFOS Induces Apoptosis in Neuroblastoma
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Cell apoptosis was evaluated using an AO (acridine orange, 100 μg/mL)/EB (ethidium bromide, 100 μg/mL) fluorescence staining assay (AO/EB, Sigma, USA). SH-SY5Y cells seeded in 12-well plates were exposed to PFOS (10, 50, 100, 150, and 200 μM) or control medium (0.1% DMSO) for 48 h, and then the cells were stained with AO/EB solution (1 : 1 v/v) for 10 min. The changes in SH-SY5Y cell nuclear morphology were immediately visualized and recorded using an inverted fluorescent microscope (Leica DM IL, German).
7
Apoptotic Cell Morphology Quantification
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The cells were treated with the test compound at its IC50 concentration and incubated for 24 h in a CO2 incubator at 37 °C. The cells were removed by trypsinization and collected by centrifugation, including the non-adherent cells. The cell pellet was re-suspended in the medium and cell suspensions (25 μL) were transferred to glass slides. Dual fluorescent staining solution containing (1 μL) 100 μg mL−1 AO and 100 μg mL−1 EB (AO/EB, Sigma) was added to the suspension and then covered with a coverslip. The morphology of the apoptotic cells was examined, and the cells were counted within 20 min using a fluorescent microscope.
8
AO/EB Dual Staining for Apoptosis
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Acridine orange/ethidium bromide (AO/EB; Sigma, St Louis, MO, USA) dual staining was done to detect the change in cell morphology. After treatment with 1.0 μM of either GA or GA-TAT for 24 h, the EJ cells were collected by trypsinization and centrifugation. The cells were washed with PBS and stained with 100 μg/mL AO and 100 μg/mL EB at 4°C in the dark for 10 min. Morphological changes, including nuclear condensation and cell shrinkage, were assessed using a fluorescence microscope (Olympus, Tokyo, Japan). The percentage of apoptotic cells was calculated by the following formula: apoptotic rate (%) = number of apoptotic cells/the total number of cells counted.
9
Cytotoxicity Evaluation of CCh on hADSCs
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The cytotoxicity effects of CCh on hADSCs viability and proliferation were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Sigma–Aldrich) assay. Briefly, 104 cells were cultured in a 96-well plate and followed by exposing different concentrations of CCh. On the 1st, 3rd, and 5th days of treatment, 100 μl of 5 mg/ml MTT solution (10 μl MTT and 90 μl DMEM) was poured into wells followed by a 4-h incubation at 37 °C and 5% CO2. Then MTT was removed and wells were treated with 100 μl DMSO (Merck, Germany) to dissolve formazan crystals. The cell viability of hADSCs was measured through absorbance at 570 nm in a microplate reader (BioTek Instruments, USA).
Additionally, to investigate the cell viability of hADSCs qualitatively, acridine orange/ethidium bromide (AO/EB, Sigma) staining was performed. At 1, 3, and 5 days after treatment, hADSCs were followed by an incubation at 37 °C with AO/EB (1 μl staining solution contains 100 g/mL AO and 100 g/mL EB) for 30 min. Finally, cells stained with AO/EB were investigated under a fluorescence microscope (Leica Inc., Foster City, CA).
Additionally, to investigate the cell viability of hADSCs qualitatively, acridine orange/ethidium bromide (AO/EB, Sigma) staining was performed. At 1, 3, and 5 days after treatment, hADSCs were followed by an incubation at 37 °C with AO/EB (1 μl staining solution contains 100 g/mL AO and 100 g/mL EB) for 30 min. Finally, cells stained with AO/EB were investigated under a fluorescence microscope (Leica Inc., Foster City, CA).
10
Synthesis and Evaluation of Rice Husk-Based Drug Delivery System
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The Rice husk used in this research was obtained from the Guangdong Academy of Agricultural Science. Cetyl trimethyl ammonium bromide, sulfuric acid, ammonia solution 25%, hydrochloric acid, Sodium hydroxide, and Dimethyl sulfoxide (DMSO) were obtained from Aladdin (Shanghai, China). Triethoxyvinylsilane, acrylic acid, potassium persulfate, and n-isopropyl acrylamide were obtained from Chemsoon Co., Ltd. (Shanghai, China). Irinotecan (SN-38) was used as received without further purification. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), fetal bovine serum (FBS), 1% penicillium-streptomycin antibiotic solution, and DMEM medium, and DMEM were purchased from Thermo Fisher Scientific. AO-EB and nuclear DAPI stain were obtained from Sigma-Aldrich (USA). All other chemicals were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).
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