Cell apoptosis was analyzed at 48 hours after drug treatment [11 (link)]. Pro-survival protein Bcl-2 was determined by Western blot. A measurement of the extent of apoptosis was carried out using an annexin V detection kit (Invitrogen, ThermoFisher, Waltham, MA, USA) to label cell surface phosphatidylserine of apoptotic cells. Briefly, treated cells were trypsinized and washed twice with phosphate-buffered saline, then suspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2). To each 100 μl cell suspension was added 5 μl annexin V conjugated to fluorescein isothiocyanate (FITC), cells were incubated at room temperature for 30 min. Stained cells were analyzed immediately by flow cytometry. To further monitor the status of cell apoptosis, T-DM1 treated cells were live stained with annexin V and propodium iodide (PI) together, co-staining images were taken by inverted fluorescence microscope. Annexin V stained cells with PI positive staining were excluded as necrotic cells, apoptotic cells with only annexin V staining were counted and divided by total cells as the percentage of apoptosis. Pan-caspase inhibitor Z-VAD-FMK (Enzo, Farmingdale, NY, USA) with working concentration 20 μM was introduced together with T-DM1 for rescue experiment, cell viability was determined as described in cell viability assay.
Annexin 5 detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Annexin V detection kit is a laboratory instrument used to detect and measure the presence of Annexin V, a protein involved in cellular processes such as apoptosis (programmed cell death). The kit provides the necessary reagents and protocols to perform Annexin V-based assays in a research setting.
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Lab products found in correlation
Pan caspase inhibitor z vad fmk, by Enzo Life Sciences (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
H3k27me3, by Abcam (1 mentions)
Facscan cytometer, by BD (1 mentions)
Anti ezh2, by BD (1 mentions)
Prism 6, by GraphPad (1 mentions)
Stempro accutase cell dissociation reagent, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Cell lab quanta sc, by Beckman Coulter (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Anti il 4 11b11, by BD (1 mentions)
Anti foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Anti bcl6 k112 91, by BD (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Prism software v, by GraphPad (1 mentions)
Facsdiva 6, by BD (1 mentions)
Collagenase, by Merck Group (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
14 protocols using annexin 5 detection kit
1
Apoptosis Analysis of Drug-Treated Cells
2
Analyzing Cell Apoptosis After Drug Treatment
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Cell apoptosis was analyzed at 48 hours after drug treatment1 (link). Pro-survival protein Bcl-2 was determined by western blot as described previously. Measurement of the extent of apoptosis was carried out using an annexin V detection kit (Invitrogen, ThermoFisher, Waltham, MA, USA) to label phosphatidylserine on the apoptotic cell surface. Briefly, cells were washed once with phosphate-buffered saline, then to each well was added 5 μl annexin V conjugated to fluorescein isothiocyanate (FITC) with binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2) in the dark for 15 minutes. Stained cells were analyzed using a Zeiss Axio Observer microscope with a 10× objective. Propidium iodide (PI) was applied to annexin V-stained cells, and cells were observed under a fluorescence microscope to check for necrotic cells.
3
Flow Cytometry for Mouse and Human T Cells
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The following antibodies were used for flow cytometric assays: For mouse T cells H3K27me3 (clone: mAbcam6147) (Abcam Inc. Cambridge, MA), CD8 (clone: 53–6.7) Invitrogen, Carlsbad, CA), CD4 (clone: GK1.5) (eBioscience). For human T cells, CD4 (clone: RPA-T4) and CD8 (clone: RPA-T8) (BD Biosciences). For both human and mouse T cells, anti-EZH2 (clone: 11/EZH2) (BD Biosciences) and Annexin V detection kit (eBioscience). For chimerism testing, mouse H2Kd (clone: SF1-1.1) and CD45.2 (clone: Ly5.2, LCA) were used (BD Biosciences). All data were collected on a FACScan cytometer (BD Biosciences, Mountain View, CA) and analyzed using FlowJo 9 (Tree Star Inc, Ashland, OR).
4
Annexin-V Apoptosis Assay Protocol
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The assay was performed using Annexin-V detection kit from e-Biosciences (Grand Island, NY, USA) as per the manufacturer’s instructions as follows. Cells (0.5 × 106) were grown in six-well plates with complete growth media for 24 h until they are semiconfluent. Cells were treated with respective concentrations of FCY-302 and incubated in a CO2 incubator for 48 h. Harvested cells were washed twice with wash buffer and incubated with 0.25 μg/ml annexin V reagent in 1× binding buffer for 15 min. After a couple of washes with wash buffer, cells were resuspended back in binding buffer containing 0.5 μg/ml propidium iodide, and 10,000 events were acquired on a Guava easyCyte™ flow cytometer. Data were analyzed using InCyte software-Millipore. Early and late phase apoptotic cells were segregated with a quadriplot graph, and total percentage of apoptotic cells was represented using Graphpad Prism 6.0 software.
5
Cell Proliferation and Apoptosis Assay
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Cell proliferation was assessed using the Click-iT Plus EdU cell proliferation kit (Molecular Probes). Cells were incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 5 hr, dissociated with TrypLE Express, and fixed with ice-cold 70% ethanol for 30 min at 4°C. Next, samples were washed with 0.1% Tween 20 and incubated for 30 min at room temperature with Click-iT Plus reaction cocktail. After washing in 0.1% Tween 20, cells were incubated with 50 μg/mL propidium iodide and 10 μg/mL RNase for 30 min before acquisition. Apoptosis was assessed using the Annexin V detection kit (eBioscience) according to the manufacturer's protocol. In brief, cells were gently dissociated with StemPro Accutase Cell Dissociation Reagent (Gibco, Life Technologies) for 5 min, and washed in PBS and then in Binding Buffer provided by the kit. V450-conjugated Annexin V was incubated at 1:20 dilution for 15 min at room temperature. After washing in Binding Buffer, samples were incubated with 7-aminoactinomycin D (7-AAD) viability staining solution. Flow-cytometry acquisition was performed using FACSCanto II (BD Biosciences) and analyzed using FlowJo software.
6
Annexin V-based Apoptosis Assay
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The apoptosis assay was performed using the Annexin V detection kit (eBioscience, Austria) in accordance with the manufacturer’s instructions. Cell cycle distribution was assessed through flow cytometric measurement upon propidium iodide staining (Sigma). Flow cytometry was performed on a Cell Lab Quanta SC flow cytometer (Beckman Coulter, Vienna, Austria) or a Cytoflex (Beckman Coulter, Vienna, Austria) and analyzed with respective machine software.
7
Multifaceted Immune Cell Analysis
8
Tff1 Modulates VSMC Apoptosis
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Primary mouse VSMCs were isolated from the aorta of WT mice aged 8 weeks and cultured as described previously.[52 ] The cells were used in all experiments from passages 4–7. The cells with a density at 80–90% were treated with Tff1 (500 ng mL−1, PeproTech) and TNF‐α (100 ng mL−1) for 24 h. Then, after 24 h of incubation apoptosis was detected with an Annexin V detection kit according to the manufacturer's instructions (eBioscience). To test the Tff1 ERK‐dependent role, VSMCs were co‐cultured with Tff1 and ERK inhibitor (10 × 10−6m , ERK1/2 inhibitor 1, Selleck, SCH77298), and VSMCs were pretreated with ERK inhibitor for 1 h before the exposure to Tff1 for 24 h.
9
Annexin V-Based Apoptosis Quantification
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The early and late apoptotic cell accumulation assay was performed using an Annexin V Detection Kit from e-Biosciences (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer instructions. Briefly, the detached cells from the experimental and control groups were incubated with 0.25 µg/mL Annexin V reagent in 1x binding buffer for 15 min followed by 2 washes with a wash buffer. Cells were resuspended in the binding buffer containing 0.5 µg/mL propidium iodide, 10,000 events were acquired immediately using Beckman Coulter Novus Flow Cytometer (Indianapolis, IN, USA) and data were analyzed using Kaluza software (v1.2, Brea, CA, USA). Early- and late-phase apoptotic cells were segregated with a quadriplot graph and the total percentage of apoptotic cells was represented using Graph Pad Prism software V (v5.0, GraphPad Software, San Diego, CA, USA).
10
Apoptosis Analysis via Annexin V
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The apoptosis assay was performed using the Annexin V detection kit (eBioscience) in accordance with the manufacturer's instructions. Briefly, 1 × 106 HCEC-1CT cells were transiently transfected with 5 μg of Con or WT-PAK1 plasmid DNA as previously described. Seventy-two hours after transfection, the cells were counted, and 1 × 105 cells were resuspended in binding buffer. Five microliter of the fluorochrome-conjugated Annexin V solution was added to the cell suspension and incubated for 15 minutes at 25°C. The cells were washed in binding buffer and incubated with propidium iodide solution for 3 hours in the dark at 4°C. Flow cytometry was performed on a Cell Lab Quanta SC flow cytometer (Beckman Coulter, Brea, CA) and analyzed with Quanta Analysis software.
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