AR42J cells (1×105) were collected to detect apoptotic and necrotic indices using the APC Annexin V Kit (BD Biosciences) in conjunction with the vital dye 7-amino-actinomycin (7-AAD) (BD Biosciences). Briefly, 1×105 of AR42J cells were trypsinized, washed twice with clod PBS, then resuspended in 500 μl 1x binding buffer. Samples received 5 μl each of APC Annexin V and of 7-AAD, were gently vortexed, and incubated for 15 min at 25°C in the dark. Each sample was subsequently analyzed in triplicate by using flow cytometry (BD Biosciences).
Apc annexin 5 kit
Manufactured by BD
Sourced in United States
The APC Annexin V kit is a fluorescence-based reagent used to detect and quantify apoptosis in cells. The kit utilizes Annexin V, a calcium-dependent phospholipid-binding protein, conjugated to the fluorescent dye allophycocyanin (APC). This allows for the identification of cells undergoing early stages of apoptosis.
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Lab products found in correlation
Vybrant dyecycle violet stain, by Thermo Fisher Scientific (3 mentions)
Facsaria, by BD (3 mentions)
Slr 2 flow cytometer, by BD (2 mentions)
Facsaria flow cytometer, by BD (2 mentions)
Lsr model 2 flow cytometer, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Facscanto, by BD (2 mentions)
7 amino actinomycin 7 aad, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscan, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Apc brdu flow kit, by BD (1 mentions)
7 aad viability staining solution, by BioLegend (1 mentions)
Flow cytometry, by BD (1 mentions)
Universal probe library system, by Roche (1 mentions)
Facsaria 2, by BD (1 mentions)
Facstation, by BD (1 mentions)
Facstation, by Beckman Coulter (1 mentions)
Propidium iodide, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
20 protocols using apc annexin 5 kit
1
Apoptosis and Necrosis Quantification
2
Flow Cytometry Analyses of Apoptosis
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Flow cytometry analyses were performed using a LSR Model II flow cytometer (BD Biosciences, Oxford, UK). Antibodies used for flow cytometry were all used at a dilution of 1/200. Apoptosis was assessed using a BD PharMingen APC Annexin V kit. Propidium iodide cell cycle analyses were performed as described (Somerville et al., 2015 (link)). Throughout the study, geometric mean cell fluorescence values are used.
3
Annexin V-Based Apoptosis Assay
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Cell apoptosis was analyzed with an APC-annexin V Kit (BD Pharmingen). After 48 h of serum-starved culture, cells in each group were fully harvested and resuspended in annexin-V binding buffer. Next, cells were stained with APC-annexin V and 7-AAD Viability Staining Solution and incubated in the dark for 30 min at room temperature before being detected by a flow cytometer.
4
Apoptosis and Mitochondrial Depolarization in GFP-expressing Cells
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Analyses were performed on the green fluorescent protein (GFP)-positive cells 48 hrs after transfections with pCMV-PID1-IRES-eGFP or pCMV-IRES-eGFP control and 24 hrs after drug treatment. AnnexinV/7AAD staining was by flow cytometry using the APC AnnexinV kit (BD Pharmingen catalog #550474) according to manufacturer’s instructions. Mitochondrial depolarization was measured by flow cytometry using the MitoProbe™ DiIC1(5) Assay Kit for Flow Cytometry (Life Technology catalog #M34151). Caspase 3 cleavage staining was assessed using anti-cleaved-caspase 3-pacific blue antibody (Cell Signaling catalog #8788) by flow cytometry. Flow cytometry analysis was performed using an SLR II flow cytometer (BD Biosciences). Cell clumps and sub-cellular debris were excluded using appropriate gating on forward and side light scatter. Data were analyzed using FACSDIVA software (BD Biosciences).
5
Quantifying Apoptosis by Flow Cytometry
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Double staining of Annexin V and 7-aminoactinomycin-D (7-AAD) was carried out using a PE Annexin V Apoptosis Detection Kit (BD Pharmingen™, United States) and an APC Annexin V Kit (BD Pharmingen™, United States) according to the manufacturer’s recommendations. Briefly, cells were washed with cold phosphate-buffered saline and then resuspended in binding buffer at a concentration of 1 × 106 cells/ml. Then, 100 μl of solution (1 × 105 cells) was transferred to a tube, and 5 μl of PE Annexin V and 5 μl of 7-AAD were added. After incubation for 15 min at room temperature in the dark and the addition of 400 μl of binding buffer, flow cytometric analysis was performed (FACScan, BD Biosciences, San Jose, CA, United States) within 1 h, and the data were analyzed with FlowJo software (Treestar, Ashland, OR). PE Annexin V ( +) or APC Annexin V ( +) and 7-AAD (-) cells represent the early stage of apoptosis, whereas PE Annexin V ( +) or APC Annexin V ( +) and 7-AAD ( +) cells are in the end stage of apoptosis or are already dead.
6
Quantifying Apoptosis and Proliferation
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The APC AnnexinV kit (BD Pharmingen cat#550474) was used to assess apoptosis and the APC BrdU Flow Kit (BD Pharmingen cat#552598) was used to assess proliferation, both according to manufacturer’s instructions. Analyses were performed by flow cytometry focusing on the eGFP-expressing or FAM-labeled cells using an SLR II flow cytometer (BD Biosciences).
7
Apoptosis Assay for Rat Neural Progenitor Cells
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Rat NPCs were grown in adherent cultures in self-renewal medium. Cultures were pre-treated with indicated concentration of DMF (or DMSO control) overnight. After H2O2 treatment, the NPC cells were trypsinized, washed with 1× Annexin binding buffer and stained with APC-Annexin V kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. 7-AAD viability staining solution (BioLegend, San Diego, CA, USA) was used to determine the necrotic cells. Staining was analyzed by flow cytometry and the apoptotic cell population was calculated by the percent of Annexin V positive cells.
8
Cell Cycle and Apoptosis Analysis
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Cell samples with specific acidic treatment for 48 h were dissociated from 6-well plates and washed twice with pre-cold DPBS. For cell cycle analysis, 1 ml DPBS was used to resuspend cells after centrifugation. Cells were fixed by dropping into 4 ml pre-cold 70 % ethanol in DPBS and kept at −20 °C for at least 24 h. After that, the samples were centrifuged and washed again, followed by staining with 20 μg/ml propidium iodide (PI, Sigma–Aldrich, USA) in DPBS containing 10 μg/ml RNase for 30 min in the dark. The APC Annexin V kit (BD, USA) and PI were performed to determine cell apoptosis. According to the manufacturer's instructions, binding buffer was used to resuspend ADSCs and 100 μl of the solution containing 105 cells was transferred to a new tube. After staining with APC Annexin V and PI for 15 min in the dark, an additional 400 μl of binding buffer was added to each tube. All samples were analyzed by flow cytometry (BD, USA). Data were calculated by FlowJo and ModFit LT software.
9
Müller Cell Apoptosis Analysis
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Müller cells from different treatment groups were harvested for apoptosis analysis using an APC Annexin V kit (#561012, BD Biosciences, Franklin Lakes, NJ, USA). In brief, 1 × 10 5 Müller cells were collected and incubated with Annexin V and propidium iodide. The percentage of apoptotic cells was determined using a FACSAria flow cytometer (BD Biosciences).
10
Apoptosis Analysis of Transfected HTMCs
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After 48 h of transfection, HTMCs were subjected to apoptosis analysis using an APC Annexin V kit (#561012, BD Biosciences, USA). Briefly, the cultured cells were resuspended, and 1 × 105 cells were added to 100 μL of 1 × binding buffer, treated with 5 μL of FITC Annexin-V for 20 min, and finally with 3 μL of propidium iodide (PI) for 10 min in the dark at room temperature. Then, 400 μL of binding buffer was added to the cells, and the rate of apoptosis was detected by a FACSAria Flow Cytometer (BD Biosciences).
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