The apoptosis-inducing effect of cisplatin in OC cell lines was determined using a PE Annexin V/7-AAD apoptosis detection kit (BD Pharmingen, USA). A2780 cells were seeded in 6-well plates at a number of 2×105 cells/well. After transfection with either siRNA or plasmid for 48 h, cells were cultured with the IC50 concentration of cisplatin for another 48 h. Adhering and floating cells were subsequently collected by trypsinization and centrifugation at 3000 rpm for 5 min. After washing twice with cold PBS, cells were diluted in annexin binding buffer at a concentration of 1×106 L. Five microliters of PE Annexin V/7-AAD was added and the cells were incubated for 15 minutes at room temperature in the dark according to the manufacturer’s protocol. Stained cells were analyzed in different experimental groups using a flow cytometer (FACSCalibur, BD Biosciences).
Pe annexin v 7 aad apoptosis detection kit
Manufactured by BD
Sourced in United States
The PE Annexin V/7-AAD apoptosis detection kit is a laboratory product designed to detect and quantify apoptosis in cell populations. It utilizes Annexin V, a protein that binds to phosphatidylserine, and 7-AAD, a DNA-binding dye, to distinguish between viable, early apoptotic, and late apoptotic or necrotic cells.
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Lab products found in correlation
Facscalibur, by BD (3 mentions)
Propidium iodide, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Thermo Fisher Scientific (1 mentions)
Jc 1 mmp assay kit, by Cayman Chemical (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rnx plus solution for total rna isolation, by Sinaclon (1 mentions)
Galunisertib, by Selleck Chemicals (1 mentions)
Sd208, by Selleck Chemicals (1 mentions)
Lyse fix buffer, by BD (1 mentions)
Perm buffer, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Cflow plus analysis software, by BD (1 mentions)
Hoechst 33258 dye, by Merck Group (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Antibodies against bax and bcl 2, by Santa Cruz Biotechnology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
11 protocols using pe annexin v 7 aad apoptosis detection kit
1
Apoptosis Analysis of Cisplatin in OC Cells
2
Cytotoxicity and Apoptosis Analysis
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Roswell Park Memorial Institute medium (RPMI-1640) and fetal calf serum (FCS) were purchased from Gibco (Ashland, KY). Cell culture grade dimethyl sulfoxide (DMSO), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), trypan blue dye and propidium iodide (PI) were purchased from Sigma (St. Louis, MO). PE Annexin V/7-AAD apoptosis detection kit was obtained from BD Pharmingen™ (San Diego, CA) and JC-1 MMP assay kit from Cayman Chemical (Ann Arbor, MI). RNX-Plus solution for total RNA isolation was obtained from Sinaclon (Tehran, Iran). SYBR Premix Ex Taq II and high-capacity cDNA reverse transcription kit were provided by Applied Biosystems (ABI, Foster City, CA).
3
Intracellular Phosphorylated SMAD Analysis
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For intracellular staining of phosphorylated SMAD (pSMAD), cells were fixed in 1 mL of Lyse-Fix buffer (BD Bioscience, San Jose, CA, USA) for 10 min at 37 °C and then permeabilized on ice in permbuffer (BD Bioscience) for 30 min. Samples were washed with PBS containing 0.5% BSA and stained with 2 µL antibody per sample. For the assessment of inhibitor function, cells were seeded in 24-well plates at a density of 0.35 × 106/mL and treated with 5 ng/mL TGF-β in the presence or absence of varying concentrations of inhibitors. Apoptosis was measured with a PE-AnnexinV/7AAD apoptosis detection kit (BD Bioscience) according to the manufacturer’s instructions after overnight treatment with TGF-β (5 ng/mL; Immunotools), galunisertib (3.5 µM) or SD208 (0.5 µM) (Selleckchem, Housten, TX, USA) in the presence or absence of 100 µM ARA-C. For cell cycle staining, 0.25 × 106 pelleted cells were resuspended in 250 µL PBS/EDTA (1 mM). Samples were fixed by the addition of 750 µL absolute ethanol and overnight incubation at 4 °C. Prior to staining, cells were washed in PBS and treated with 100 µg/mL RNAse A in a total volume of 475 µL PBS/EDTA for 20 min at 37 °C. Propidium iodide (Sigma) was added to a final concentration of 50 µg/mL to distinguish between cell cycle phases in terms of cellular DNA content.
4
Quantifying Viral-Induced Apoptosis by Flow Cytometry
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Cell apoptosis was assessed using a PE-Annexin-V/7-AAD apoptosis detection kit (BD Pharmingen, San Jose, CA) and the apoptotic rate was analyzed by a flow cytometry on FACS Calibur. Data were analysed using CellQuest software version 2.0. A total of 5×105−1×106 cells/well were seeded in 6-well plates for 24 h at 37°C. Following 96-h of viral transfection, as described above, cells were harvested and washed with PBS 2–3 times at 37°C for 5 min. Subsequently, cells were resuspended at a density of 1×106 cells/ml, stained with Annexin V-PE and counterstained with 7-AAD in binding buffer (included in kit) at room temperature for 15 min. The apoptotic cells were measured using a flow cytometer with 488 nm excitation and 578 nm emission for Annexin V-PE detection, and 488 nm excitation and 647 nm emission for 7-AAD detection.
5
Cell Cycle Analysis by Flow Cytometry
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Cells were plated at a density of 1.5 × 106 per 60-mm dish and allowed to grow for 24 h, after which the medium was changed to serum-free medium. After 24 horrs of serum starvation, cells were released to regular completed medium for another 24 hours. Then cells were harvested by trypsinization, washed twice with PBS, and pelleted by centrifugation for 5 min at 500 × g. Cells were then resuspended in PBS, fixed with 75% cold ethanol overnight at 4°C, washed twice with PBS, and subsequently resuspended in 1 ml of 50 μg propidium iodide solution with 50μg RNase A and incubated for 30 minutes avoid of light before analysis. Cells were next filtered through a 100-mesh filter, and a total of 20,000 stained nuclei were analyzed with ACCURI C6 flowcytometer (BD Biosciences, San Jose, CA, USA) and CFlow Plus analysis software. Cell death was determined by using PE AnnexinV/7 AAD Apoptosis Detection Kit (BD Biosciences) following manufacturer’s instructions. Data were presented as mean ± SEM from triplicates.
6
Apoptosis Quantification in Treated Cells
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Control or 7 day AA/BGP treated cells were harvested with StemPro Accutase Cell Dissociation Reagent (Gibco GE Healthcare). Apoptosis analysis was performed using the PE Annexin-V/7-AAD Apoptosis Detection Kit (BD Biosciences Pharmingen) or multicaspase fluorogenic substrate (SR) (Guava-Merck Millipore, USA, Billerica) according to the manufacturer’s instructions. Briefly, cells were washed in PBS and suspended in binding buffer for staining with PE Annexin-V and 7-AAD (7-aminoactinomycin D) at room temperature for 15 min in the dark. The cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson). The signal obtained from cells stained with annexin-V or 7-AAD alone was used for fluorescence compensation. To measure caspase activation the cells were incubated with the multicaspase substrate for 1 h under cell culture conditions followed by staining with 7-AAD. Fluorescence was determined using the microplate reader in a Guava easyCyte 8HT Benchtop Flow Cytometer (Guava-Merck Millipore) and acquired using the Guava Caspase Software Module. Cells stained with SR or 7-AAD alone were used for fluorescence compensation.
7
TMT-Mediated Signaling Pathway Modulation
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TMT (>98% purity) was obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Dulbecco's modified Eagle's medium (DMEM) for cell culture was purchased from Hyclone. LBP (active ingredient > 31%) was provided by Shanghai Kangzhou Fungi Extract Co. (Shanghai, China). Antibodies against GSK-3β, p-GSK-3β (Ser9), Akt, p-Akt (Thr308), CyclinD1, C-Myc, GAPDH, and PI3K inhibitor LY294002 were acquired from Cell Signal Technology, Inc. (Beverly, MA). Antibodies against Gli1 and Shh were obtained from Abcam Inc. Antibodies against Bcl-2 and Bax were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Shh inhibitor GDC-0449 was obtained from Selleckchem, Inc. Reactive oxygen species (ROS) and caspase-3 activity assay kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Malondialdehyde (MDA) and superoxide dismutase (SOD) kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). PE-Annexin-V/7-AAD apoptosis detection kit was acquired from BD Pharmingen, Inc. (San Diego, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst33258 dye were provided by Sigma Co.
8
Cisplatin-induced Apoptosis Evaluation
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Cells were treated with cisplatin. After 24 h, cells were collected, and apoptotic cells were assessed by TUNEL Apoptosis Detection kit (Vazyme, Cat. #A113) or PE-Annexin V/7-AAD Apoptosis Detection kit (BD Biosciences, Cat. #559763) according to the manufacture’s instruction.
9
Cell Death Assay with IFN-γ and Cisplatin
10
Annexin V and 7-AAD Apoptosis Assay
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The PE Annexin V/7-AAD Apoptosis Detection Kit (BD Biosciences) was used to detect the extent of apoptosis. After treating 143B and U2OS cells with different concentrations of ISO, the cells were washed twice with PBS and resuspended in 1X binding buffer at a density of 1 × 10 * 6 cells/ml. A total of 5 μL of PE annexin V and 5 μL of 7-AAD were added to 500 μL of this solution (1 × 10 × 5 cells) and incubated for 15 min at RT (25 °C) in the dark. Flow cytometry (Beckman Coulter, USA) was used to analyze the level of apoptosis.
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