The pro-apoptotic activity of [10]-gingerol was analysed by flow cytometry with the PE-Annexin-V Apoptosis Detection Kit (Becton, Dickinson, Franklin Lakes, NJ, USA) or using the TUNEL assay kit (Promega Corporation, USA) according to the manufacturer's instructions and as described in Supplementary methods.
Pe annexin 5 apoptosis detection kit
Manufactured by BD
Sourced in United States, China, United Kingdom, Canada
The PE Annexin V Apoptosis Detection Kit is a laboratory product used for the detection and analysis of apoptosis, a programmed cell death process. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a cellular membrane component exposed during apoptosis. The kit also includes propidium iodide, a dye that stains the nuclei of cells with compromised cell membranes. This combination of reagents allows for the identification and differentiation of viable, early apoptotic, and late apoptotic or necrotic cells through flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (31 mentions)
Facscalibur, by BD (31 mentions)
Cellquest pro software, by BD (15 mentions)
Facscan, by BD (12 mentions)
Facscanto 2, by BD (10 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (9 mentions)
Flow cytometry, by BD (8 mentions)
Cellquest software, by BD (8 mentions)
Accuri c6, by BD (7 mentions)
Pi rnase staining buffer, by BD (7 mentions)
Facsdiva software, by BD (6 mentions)
Accuri c6 flow cytometer, by BD (6 mentions)
Propidium iodide, by Merck Group (6 mentions)
Facscan flow cytometer, by BD (5 mentions)
Apc brdu flow kit, by BD (5 mentions)
Cycletest plus dna reagent kit, by BD (4 mentions)
Facsaria, by BD (4 mentions)
Facscanto 2 flow cytometer, by BD (4 mentions)
Lsrfortessa, by BD (4 mentions)
Facscanto, by BD (4 mentions)
445 protocols using pe annexin 5 apoptosis detection kit
1
Apoptotic Activity of [10]-Gingerol
2
Annexin V Apoptosis Assay in A2780 Cells
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Herein, 3 × 105 A2780 cells were grown as indicated by the supplier for indicated
time points. Annexin V analysis was performed using PE-Annexin V Apoptosis
Detection Kit from Becton–Dickinson (Franklin Lakes, NJ) according
to the manufacturer’s protocol using FACS Canto II from Becton–Dickinson
(Franklin Lakes, NJ) and BD FACS DIVA software.
time points. Annexin V analysis was performed using PE-Annexin V Apoptosis
Detection Kit from Becton–Dickinson (Franklin Lakes, NJ) according
to the manufacturer’s protocol using FACS Canto II from Becton–Dickinson
(Franklin Lakes, NJ) and BD FACS DIVA software.
3
Apoptosis Detection in Normoxia and Hypoxia
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The PE Annexin V Apoptosis Detection Kit (Becton Dickinson) was used to detect apoptosis in cells treated with CisPt at IC25 dose (5.9 µM), Res at IC25 dose (63 µM), and CisPt+Res (at the same concentrations) in normoxia and hypoxia, as described in Section 4.4 : Establishment of Hypoxic Culture Conditions and Experimental Outline. After 48 h incubation with drugs, cells were harvested and stained according to the manufacturer’s protocol. Then, the samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson), equipped with 355 nm, 405 nm, 488 nm, and 640 nm lasers. Data were analyzed using FCS Express 4 Flow software.
4
Quantifying Apoptosis by Flow Cytometry
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Apoptotic cells were assessed using the PE Annexin V apoptosis detection kit (Becton, Dickinson and Company, NJ, USA). Cells in different groups were digested and resuspended in 100 µL of binding buffer containing 5 µL of PE Annexin V and 5 µL of 7-Amino-Actinomycin for 15 min at room temperature. Then, 400 µL of binding buffer was added for 1 h of incubation. Finally, cells were analyzed by flow cytometry on a FACSCalibur flow cytometer (Becton-Dickinson, Franklin-Lakes, NJ, USA).
5
Cytotoxicity and ROS Evaluation of SNP
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Sodium nitroprusside dihydrate (Na2[Fe(CN)5NO]·2H2O, SNP, ≥99%) (SNP), silver nitrate (AgNO3), and anhydrous methanol (99.8%) were purchased from Sigma-Aldrich (St. Louis, USA). A Millipore Milli-Q Biocell A10 water purifying system was used to prepare ultrapure water. A2780, A2780cis, U-87 MG (Sigma-Aldrich, St. Louis, MO, USA), MDA-MB-231 (Cell Biolabs, CA, USA) SK-OV-3, MCF-7, MRC-5, W1-38 (ATCC, Manassas, VA, USA) cells were cultured according to the manufacturers’ instruction. LysoTracker Green DND-26, Hoechst 33342, and rhodamine B were purchased from (ThermoFisher Scientific, Waltham, MA, USA). CellTiter-Glo® from (Promega, Madison, WI, USA), PE-Annexin V Apoptosis Detection Kit from (Becton-Dickinson Franklin Lakes, NJ, USA). ROS-Glo™ H2O2 luminescence assay from (Promega, Madison, WI, USA), DAX-J2 PON Green Kit was purchased from (AAT Bioquest, Sunnyvale, CA, USA), FluorSave™ reagent (Millipore, Burlington, MA, USA). qPCR Primers from (Integrated DNA Technologies, Bologna, Italy).
6
Cell Viability and Death Assays
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Cell viability was determined by Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) through the reagent WST-8 as described previously [24 (link)]. Treated cells were incubated in 10% CCK-8 that was diluted in normal culture medium at 37°C until the visual color conversion occurred. Optical density (OD) values were assessed at 450 nm using a full-wavelength Varioskan Flash. Cell death was determined by phycoerythrin (PE) annexin V in conjunction with 7-aminoactinomycin (7-AAD) staining by using the PE Annexin V Apoptosis Detection Kit (Becton-Dickinson, San Jose, CA) according to the manufacturer's protocol.
7
Apoptosis and Cell Cycle Analysis of 5Z7O Treatment
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Cells treated with a specified concentration of 5Z7O or vehicle for 48 hours and then assayed for apoptosis and cell cycle progression. Apoptosis detection was performed using PE Annexin V apoptosis detection kit (Becton-Dickinson #559763). Cells were treated with fixation/permeabilization solution kit (Becton-Dickinson #554714) before staining with anti-Ki-67 FITC (Thermo Fisher #11-5698-82) and 7AAD for cell proliferation and DNA content, respectively. Flow cytometry analysis was conducted using FACS Canto (Becton-Dickinson Bioscience) and FlowJo software (TriStar).
8
Molecular Mechanisms of Neurodegeneration
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The rotenone, puromycin, 3-Methyladenine (3-MA), Dorsomorphin, 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and retinoic acid (RA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Bafilomycin A1 was obtained from Aladdin (Shanghai, China). The PE Annexin V Apoptosis Detection Kit was purchased from the Becton-Dickinson Company (Franklin lakes, NJ, USA). The GSH Assay Kit and SOD Assay Kit were provided by the Nan-Jing Jiancheng Biocompany (Nanjing, Jiangsu, China). The MMP Assay Kit with JC-1 was from Beyotime (Haimen, Jiangsu, China). The antibodies were purchased from the following companies: alpha-synuclein from the Becton-Dickinson Company (Franklin lakes, NJ, USA); LKB1, p-LKB1 (Ser428), AMPK, p-AMPK (Thr172), and Beclin 1 from Cell Signaling Technology (Danvers, MA, USA); LC3 from Sigma-Aldrich (St. Louis, MO, USA); mTOR (S2442) and p-mTOR (S2448) from Bioworld (St. Louis Park, MN, USA); β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); goat anti-mouse IgG secondary antibody and goat anti-rabbit IgG secondary antibody (both peroxidase conjugated) were purchased from Signalway Antibody (College Park. MD, USA). A goat anti-mouse IgG (Cy3-conjugated) was purchased from Zhuangzhi Biotechnology (Xi’an, China).
9
Apoptosis Detection by Flow Cytometry
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The cells were labeled with dyes in PE Annexin V Apoptosis Detection Kit (Becton Dickinson, USA), and then analyzed by flow cytometer (BD FACS Calibur System, Franklin Lakes, NJ, USA). Cells population were classified based on whether it was labeled with neither Annexin V (AV) nor 7-AAD (viable cells), AV alone (apoptotic cells), 7-AAD alone (necrotic cells), or both AV and 7-AAD (late apoptotic cells), as described previously 32 .
10
Quantifying Apoptosis via Flow Cytometry
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