DCZ3301 (purity > 98%) was synthesized by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. This compound has been patented and the relevant patent number is 2016102204055 recorded by State Intellectual Property Office Of The P.R.C. DCZ3301 was dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) at a concentration of 16 mmol/L (16 mM) and stored at -20℃ until use. IL-6 and VEGF were purchased from R&D Systems (Minneapolis, MN, USA). Human CD138 MicroBead was obtained from Miltenyi (Miltenyi Biotec GmbH, Germany). Antibodies for caspase 3, 8, and 9, PARP, ERK1/2, AKT, STAT3, phospho(p)-ERK1/2, p-AKT, p-STAT3, β-actin were purchased from Cell Signaling Technology; Cdc25C, CDK1, Cyclin B1, IKBα, p-IKBα(Ser32), p-p65(S536) were from Abcam. Z-VAD-FMK was provided by Selleck Chemicals (Houston, TX, USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo. Annexin V-FITC and PI detection kit was purchased from BD Pharmingen (San Diego, CA). Mitochondrial membrane potential assay kit with JC-1 was obtained from Beyotime Institute of Biotechnology.
Annexin 5 fitc and pi detection kit
Manufactured by BD
Sourced in United States
The Annexin V-FITC and PI detection kit is a laboratory reagent used for cellular apoptosis analysis. It contains Annexin V labeled with the fluorescent dye FITC and the DNA-binding dye Propidium Iodide (PI). This kit allows for the identification and quantification of apoptotic and necrotic cells through flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Cyclin b1, by Abcam (1 mentions)
Phospho perk1 2, by Cell Signaling Technology (1 mentions)
Human cd138 microbead, by Miltenyi Biotec (1 mentions)
Cdc25c, by Abcam (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Z vad fmk, by Selleck Chemicals (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Facsdiva software v7, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
5 protocols using annexin 5 fitc and pi detection kit
1
Synthesis and Characterization of DCZ3301
2
Annexin V-FITC/PI Apoptosis Assay
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We stained cells using Annexin V-FITC and PI Detection Kit (BD Pharmingen, USA) prior to the analysis with flow cytometry. Briefly, cells were seeded in 35-mm Petri dishes at a density of 1 × 106 cells per well. The cells were cultured in 2 mL phenol red-free medium containing 10% FBS, and necroptosis was then induced as described above. Then, the foam cells were measured at suitable time points according to the manufacturer. The results were analysed using BD FACSDiva Software v7.0 (Becton-Dickinson, USA). For proper statistical analysis, more than 10000 cells per group were counted, and each assessment was repeated three times.
3
Annexin V-FITC Apoptosis Assay in RWPE-1 and BPH-1 Cells
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Annexin V-FITC and PI Detection Kit (BD Biosciences, NJ, USA) was used to stain RWPE-1 and BPH-1 cells to determine cell apoptosis. RWPE-1 or BPH-1 cells at a density of 2 × 105 were treated with finasteride/DMSO and mimics/inhibitors. They were then harvested, resuspended in 100µL flow cytometry binding buffer, and then stained with 5µL Annexin V/FITC and 5µL PI following the manufacturer’s instructions. Apoptosis was determined using flow cytometry (BD FACSCanto™ II, NJ, USA).
4
Ultrasound-Mediated Sonodynamic Therapy
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Cells were seeded (8 × 104 cells/well) on circular coverslips placed in a 24-well plate. Cells were randomly divided into four groups (Control, DVDMS, US, and SDT) as mentioned above. The cells in DVDMS and SDT groups were incubated with DVDMS (5 µg/mL) in the dark for 6 h. Then, the cells in US and SDT groups were exposed to ultrasound for 3 min. After treatments, cells were cultured for 2 h before flow cytometry analyses. Cellular apoptosis and necrosis were analyzed with an Annexin V-FITC and PI detection kit (BD Biosciences, San Diego, CA, USA). In brief, the cells were harvested and suspended in a flow tube using 200 µL 1× binding buffer. To adjust the fluorescence compensation, a blank control group (without staining) and two groups stained with PI or FITC solution separately were prepared. The cells in the four groups (Control, DVDMS, US and SDT) were stained with both FITC and PI solution. After staining in the dark, the apoptosis and necrosis of cells were immediately evaluated with flow cytometry (BD Accuri C6 Plus, BD Biosciences, Franklin Lakes, NJ, USA) using the FL-1 filter (excitation 488 nm, emission 525 nm) and FL-2 filter (excitation 488 nm, emission 590 nm).
5
Annexin V and PI Staining of Macrophages
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Before the analysis with flow cytometry, macrophages and foam cells were stained cells by using the Annexin V-FITC and PI Detection kit (BD Pharmingen; BD Biosciences). In brief, the cells were trypsinized and resuspended at a density of 106/ml. After centrifugation, the pellet was washed twice with ice-cold PBS and was suspended in binding buffer. The cells were maintained in the dark at room temperature for 15 min after incubation with FITC-labelled Annexin V and PI (Molecular Probes; Thermo Fisher Scientific, Inc.), and the cells were analysed using flow cytometry. More than 1×104 cells per group were counted and each assessment was repeated three times to get a proper statistical analysis.
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