Phosphatidylserine translocation in MOLT-4 cells treated with Pd@W.tea NPs was determined by the Annexin-V-FITC/propidium iodide (PI) apoptosis detection kit (Sigma-Aldrich Co.).21 (link) In brief, cells treated with 0.006 μM Pd@W. tea NPs for 12, 24, or 48 h were harvested, washed with PBS twice, resuspended in binding buffer, and incubated with Annexin V-FITC and propidium iodide solutions at room temperature in the dark for 30 min. Apoptosis was determined by flow cytometry using FACS Calibur (BD Biosciences, San Jose, CA, USA) with 15,000 ungated cells.
Annexin 5 fitc propidium iodide apoptosis detection kit
Manufactured by Merck Group
Sourced in United States
The Annexin V-FITC/propidium iodide (PI) apoptosis detection kit is a laboratory assay used to detect and quantify apoptosis, a form of programmed cell death, in cell samples. The kit utilizes fluorescent-labeled Annexin V and propidium iodide to distinguish between viable, early apoptotic, and late apoptotic/necrotic cells.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (1 mentions)
Human insulin, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Cleaved parp, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Bcl 2, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium nutrient mixture f 12 dmem f 12, by Caisson (1 mentions)
Lsrfortessa, by BD (1 mentions)
Prism version 6, by GraphPad (1 mentions)
Facscan flow cytometry, by BD (1 mentions)
Pi staining solution, by Merck Group (1 mentions)
Facsc, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Cellquest version 3, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
11 protocols using annexin 5 fitc propidium iodide apoptosis detection kit
1
Apoptosis Detection in MOLT-4 Cells
2
Cell Culture Reagents and Assays
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Eagle’s Minimum Essential Medium (EMEM), Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12) were purchased from Caisson Lab (Smithfield, UT, USA). The cell culture products, including Trypsin-EDTA, fetal bovine serum (FBS), penicillin, and streptomycin, were purchased from Gibco Thermo Fisher Scientific (Waltham, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), and standard Penta-galloyl-β-D-glucose hydrate (PGG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The additional growth factors for cell culture, such as human insulin, epidermal growth factor, basic fibroblast growth factor, and hydrocortisone, were obtained from Sigma-Aldrich (St. Louis, MO, USA). The annexin V-FITC/propidium iodide (PI) apoptosis detection kit was also purchased from Sigma-Aldrich (St. Louis, MO, USA). PMSF and cocktail protease inhibitors were purchased from Hi Media Laboratories (Marg, Mumbai, India). Primary antibodies against total Akt, phosphorylated Akt (Ser473), total STAT3, phosphorylated STAT3 (Tyr705), cleaved-caspase 3, Bcl2, cleaved-PARP, and GAPDH, as well as horseradish peroxidase-labeled secondary antibodies, were purchased from Merck (Merck, Darmstadt, Germany).
3
Quantifying Apoptosis in SH-SY5Y Cells
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Cell apoptosis was measured by flow cytometry through an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Sigma) according to the manufacturer's protocols. Briefly, treated SH-SY5Y cells were collected and incubated with Annexin V-FITC and PI for 20 min in the dark after being washed with PBS. The positive cells were examined using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
4
Annexin V-FITC/PI Apoptosis Assay
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Cellular apoptosis was determined using Annexin V-FITC/Propidium Iodide (PI) apoptosis detection kit (Sigma Aldrich, USA), according to the manufacturer’s instructions, and was analyzed using flow cytometry, to determine the the percentage of apoptotic cells after treatments with H2O2 and RECA. Cells were treated as stated above. Then, the cells were harvested, incubated with Annexin V-FITC and Propidium Iodide (PI), and analyzed at 525 nm for FITC and 630 for PI with a flow cytometer (LSR Fortessa; BD Biosciences, USA). Early apoptotic cells were defined as those cells that expressed only Annexin V/FITC. Meanwhile, late apoptotic cells expressed both Annexin V/FITC and PI. Dead cells expressed only PI. Apoptosis assay was run at least twice, with technical triplicates. The fluorescence intensity of 104 cells was acquired and analyzed using FACs Diva Software. For statistical analysis, one-way ANOVA was used (GraphPad Prism version 6).
5
Apoptosis and Cell Cycle Analysis
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Cells with treatment or transfection were cultured for 48 h and collected by Trypsin digestion. Cells were then washed with PBS and used for apoptosis detection using the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Sigma). In brief, cells were suspended in binding buffer and then treated with 5 µL Annexin V-FITC and 10 µL PI. Cells were incubated in the dark for 15 min, and cell apoptosis was analyzed using a FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
Cells with treatment or transfection were cultured for 24 h and collected by Trypsin digestion. Cells were washed with PBS and fixed in 70% ethanol at 4℃ overnight. Next, cells were washed with PBS and stained with PI/RNase A working buffer (BD Biosciences) in the dark for 15 min. Cell cycle distribution at various phases was determined using a FACScan flow cytometry (BD Biosciences).
Cells with treatment or transfection were cultured for 24 h and collected by Trypsin digestion. Cells were washed with PBS and fixed in 70% ethanol at 4℃ overnight. Next, cells were washed with PBS and stained with PI/RNase A working buffer (BD Biosciences) in the dark for 15 min. Cell cycle distribution at various phases was determined using a FACScan flow cytometry (BD Biosciences).
6
Cell Cycle and Apoptosis Analysis
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The aforementioned cells were incubated in 6-well plates. The cells were collected following 48 h of culture, washed twice with pre-cooled D-Hanks solution, and fixed with 70% ethanol overnight at 4˚C. Each group was centrifuged at 1,000xg for 5 min, washed with D-Hanks solution, stained with 1 ml PI staining solution (Sigma-Aldrich; Merck KGaA), and incubated at room temperature in the dark for 30 min. Cell proliferation was analyzed by flow cytometry (MilliporeSigma, Guaveasy Cyte HT).
The apoptotic rate was evaluated using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA) according to the instructions provided by the manufacturer. The cells were seeded into 6-well culture plates (4x105 cells). Following treatment, the cells were collected, washed with PBS, and resuspended in 500 μl binding buffer. Subsequently, 5 μl Annexin V-FITC and 5 μl PI were added to the buffer and incubated at room temperature for 15 min in the dark. The cells were analyzed by flow cytometry (MilliporeSigma).
The apoptotic rate was evaluated using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA) according to the instructions provided by the manufacturer. The cells were seeded into 6-well culture plates (4x105 cells). Following treatment, the cells were collected, washed with PBS, and resuspended in 500 μl binding buffer. Subsequently, 5 μl Annexin V-FITC and 5 μl PI were added to the buffer and incubated at room temperature for 15 min in the dark. The cells were analyzed by flow cytometry (MilliporeSigma).
7
Annexin V-FITC/PI Apoptosis Assay
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Cell apoptosis was analyzed by Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit according to the manufacturer's protocol (Sigma-Aldrich, Sigma Chemical Co., St. Louis, MO, USA). Briefly, H1299 cells were seeded in 6-well plates at a density of 1 × 104 cells/well and then treated with ZSD (4 mg/mL) for 12, 24, and 48 h, respectively, or different doses of ZSD. Then, cells were collected and centrifuged and resuspended in binding buffer. Afterwards, 5 μL Annexin V-FITC and 5 μL PI were added at room temperature for 15 min. Finally, apoptosis cells were analyzed by a flow cytometer (FACSC, BD Instruments Inc., USA) and FlowJo 7.1.0 software (Tree Star, Ashland, OR, USA).
8
Quantifying Apoptosis in Transfected and Hypoxic Cells
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Apoptosis rate of transfected and hypoxia-treated SNU-387 and Huh7 cells was evaluated using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocols. Briefly, the cells were collected and resuspended in binding buffer and labeled with 5 µl Annexin V-FITC and 10 µl PI in the dark at room temperature for 15 min. The apoptotic rate (early + late apoptotic cells) was analyzed using a FACScan flow cytometer (Becton-Dickinson and Company) with Cell Quest software (version 4.0.2, Becton-Dickinson and Company).
9
Quantifying CD14+ PBMC Apoptosis
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CD14+PBMC cell apoptosis was determined using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA) on a flow cytometer (BD Biosciences). Briefly, CD14+PBMCs were collected by after centrifugation at 1,000 x g for 5 min at 4˚C and resuspended in 500 µl binding buffer. Subsequently, CD14+PBMCs were stained with Annexin V-FITC (100 µl) for 15 min and PI (10 µl) for 5 min in the dark. The cell apoptotic rate was quantified on a flow cytometry and analyzed by CellQuest version 3.3 (BD Biosciences).
10
Annexin V-FITC/PI Apoptosis Assay
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Apoptosis analysis was also assessed with Annexin V-FITC/propidium iodide apoptosis detection kit (Sigma–Aldrich, U.S.A.) using LSR II flow cytometry (BD, U.S.A.). Cells were plated in a 48-well plate at a density of 0.5 × 106 per well. After incubation for 48 h post-transfection, cells were harvested and incubated with 10 μl Annexin V-FITC and 5 μl PI Staining Solution. Flow cytometry data were analyzed using CellQuestPro software (BD, U.S.A.).
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