The cell cycle was analyzed using a Cell Cycle Detection Kit (BB-4104, BestBio, Shanghai) according to the manufacturer’s instructions. The cells were harvested by centrifugation and then fixed in 75% cold ethanol overnight after washing three times with PBS. Each sample was washed and resuspended in 500 μl of propidium iodide (PI) working solution for 30 to 60 min in the dark before detection using a FACSCalibur flow cytometer (Becton, Dickinson and Company, USA). The percentage of cells was further analyzed with ModFit LT software. Cell apoptosis was measured using an Annexin V-FITC/PI staining kit (BB-4101, BestBio, Shanghai) according to the manufacturer’s guidelines. The cells were counted and adjusted to 1 × 106 cell/ml. Then, a 2 ml aliquot of the cells was inoculated into each well of a 6-well plate, and the cells were allowed to grow for 24 h prior to drug treatment. Flow cytometry was performed using a FACSCalibur flow cytometer (Becton, Dickinson and Company, USA) and processed using CellQuest Pro analysis software. The experimental methods used for flow-cytometric analysis in our study can be found in previous reports [15 (link), 16 (link)].
Annexin 5 fitc pi staining kit
Manufactured by BestBio
Sourced in China
The Annexin V-FITC/PI staining kit is a laboratory product used for the detection and quantification of apoptosis in cell samples. The kit contains Annexin V conjugated with the fluorescent dye FITC and the nuclear stain propidium iodide (PI). Annexin V binds to phosphatidylserine, which is externalized during the early stages of apoptosis, while PI stains DNA in cells with compromised cell membranes, indicating late-stage apoptosis or necrosis. The kit provides a simple and reliable method to distinguish between viable, early apoptotic, and late apoptotic/necrotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (2 mentions)
Cell cycle detection kit, by BestBio (1 mentions)
2 7 dichlorofluorescin diacetate dcfh da, by Merck Group (1 mentions)
Bicinchoninic acid bca protein assay kit, by Sangon (1 mentions)
Hrp conjugated goat anti mouse igg, by ZSGB-BIO (1 mentions)
Hrp conjugated goat anti rabbit igg, by ZSGB-BIO (1 mentions)
N acetyl l cysteine nac, by Beyotime (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
Antioxidant capacity assay kit, by Nanjing Jiancheng (1 mentions)
Glutathione peroxidase assay kit, by Nanjing Jiancheng (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Sod activity assay kit, by Nanjing Jiancheng (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Anti beclin1 antibody, by Abcam (1 mentions)
Dimethylsulfoxid, by Merck Group (1 mentions)
Anti mnsod antibody, by Abcam (1 mentions)
Anti sirt3 antibody, by Abcam (1 mentions)
Anti lc3a b antibody, by Abcam (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
12 protocols using annexin 5 fitc pi staining kit
1
Cell Cycle and Apoptosis Analysis
2
Apoptosis Quantification in HCC Cells
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To quantify cellular apoptosis, HCCLM3Vector, HCCLM3YMO+, HepG2Vector and HepG2shYMO1 HCC cells were stained with an Annexin V-FITC/PI staining Kit (Bestbio, Beijing, China) according to the manufacturer's protocol and analyzed by flow cytometry. Three independent experiments were carried out.
3
Erianin-Induced Apoptosis and Oxidative Stress
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The reagents used in this study include erianin (≥98% purity) (Tauto Biotech, Shanghai, China); 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide (MTT) (Biofroxx, Einhausen, German); Annexin V-FITC/PI staining kit (BestBio, Shanghai, China); 2’,7’-Dichlorofluorescin diacetate (DCFH-DA) (Sigma, St. Louis, MO, USA); bicinchoninic acid (BCA) protein assay kit (Sangon Biotech, Shanghai, China); N-Acetyl-L-cysteine (NAC) (Beyotime Biotechnology, Shanghai, China); antibodies against cleaved PARP, cleaved caspase-3, AKT, phosphorylated-AKT (p-AKT), mTOR, phosphorylated-mTOR (p-mTOR), JNK, phosphorylated-JNK (p-JNK), c-Jun and phosphorylated-c-Jun (p-c-Jun) (Cell Signal Technology, Boston, USA); antibodies against β-actin, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China). Erianin was dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals, Solon, OH, USA) in a 100 mM stock solution and stored in the dark at −20 °C.
4
Resveratrol Modulates Mitochondrial Function
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Resveratrol was purchased from Aladdin (Shanghai, China). NAC (N-acetyl-L-cysteine) and Dimethyl sulfoxid (DMSO) were purchased from Sigma (St. Louis, MO, USA). The MDA assay kit, SOD activity assay kit, antioxidant capacity assay kit, glutathione peroxidase assay kit and glutathione reductase assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nangjing, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (St Louis, MO, USA). A cell counting kit (CCK-8) was purchased from Biosharp (Hefei, China). Annexin V-FITC/PI staining kit was purchased from BestBio (Shanghai, China). Anti-MnSOD antibody, anti-Sirt3 antibody, anti-LC3A/B antibody and anti-Beclin1 antibody were purchased from Abcam (Beverly, MA, USA). Alexa Fluor 488-conjugated secondary antibody and 4′,6′-diamidino-2-phenylindole (DPI) were purchased from BEIJING BIOSS (beijing, China). MitoSOX Red mitochondrial superoxide indicator and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) for live-cell imaging was obtained from life technologies (San Diego, CA, USA).
5
Apoptosis Evaluation of FLSs
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The apoptotic rate of FLSs was detected by flow cytometry using Annexin V-FITC/PI staining kit (BestBio, Shanghai, China) according to the manufacturer’s protocol. Briefly, FLSs were seeded and incubated in 6-well plates and treated with above dosage and time.after that cells were digested with pancreatic enzymes, resuspended in 400 μl Annexin V binding buffer at a density of 1 × 106cells/ml and incubated with 5 μl Annexin V-FITC and 10 μl propidium iodide (PI) for 15 min at 4 °C in dark. Finally, cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences).
6
Cell Cycle and Apoptosis Analysis
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Flow cytometry was applied to detect cell cycle and apoptosis. PCa cells were stained with propidium iodide (PI) (Beyotime, C1052, Shanghai, China). Cells were washed and resuspended in cold phosphate buffered saline (PBS) and incubated in ice-cold 70% ethanol for 4 h. The cells were then centrifuged at 1500 rpm for 10 min and resuspended in propidium iodide (PI) master mix at a density of 5ⅹ105 cells/mL and incubated at 37°Cfor 30 min before analysis with flow cytometry as described. For apoptosis assays, cells were harvested as before and double stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI. Apoptotic cells were detected using Annexin V-FITC/PI staining kit (BestBio, BB-4101-3, Shanghai, China).
7
Quantifying Apoptosis with Annexin V-FITC/PI
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The rates of apoptosis were detected using the Annexin V-FITC/PI staining kit (BestBio#BB-4101) according to the manufacturer’s procedures. Briefly, cells grown in 6-well plates were harvested, washed with cold PBS and re-suspended in 1× Annexin-binding buffer at °C before the sequential addition of 5 μl Annexin V-FITC solution for 15 min and then 10 μl PI for 5 min. The cells were analyzed using flow cytometry within 1 h of staining.
8
Apoptosis Analysis by Flow Cytometry
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Flow cytometry analysis of apoptosis was analyzed using an Annexin V-FITC/PI staining kit (BestBio, Shanghai, China) according to the manufacturer’s instructions. Twenty-four hours after transfection, cells (1×104 cells/ well) were stimulated with 5μM cisplatin for 48h and then collected. After washing 3 times with with cold PBS, the cells were resuspended in binding buffer followed by staining with Annexin V-FITC for 15min and PI for 5min in darkness. 1× 104 cells were assessed on a FACS Calibur flow cytometer (BD, Bedford, MA, USA). All experiments were done in triplicate.
9
Apoptosis Evaluation of VSMCs
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Annexin V‐FITC/PI staining kit (BestBio) was employed to determine cell apoptosis of VSMCs following the protocol. Finally, cell apoptosis was assessed using flow cytometry.
10
Quantification of HMME-PDT-induced Apoptosis
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