Cal27, OSC19, and SCC25 cells were grown in 6-cm dishes to 70% confluence in 5% FBS. Cells were incubated with the desired concentration of cisplatin and/or gefitinib for 72 h. The conditioned medium from each well was collected; cell monolayers were trypsinized, resuspended in the corresponding conditioned medium, centrifuged at 3000 rpm for 3 min at 4 °C, washed once with cold PBS, and resuspended in annexin V-FITC and PI according to the manufacturer’s recommendations (Millipore). Flow cytometry was conducted in the UVA Flow Cytometry Core Facility with marker combinations as follows: cells that are viable are both annexin V and PI negative; cells in early apoptosis are annexin V positive and PI negative; cells in late apoptosis are annexin V and PI positive; necrotic/dead cells are annexin V negative and PI positive.
Annexin 5 fitc and pi
Manufactured by Merck Group
Sourced in United States
Annexin V-FITC and PI are fluorescent labeling reagents used for the detection and quantification of apoptotic cells. Annexin V binds to phosphatidylserine exposed on the cell surface during apoptosis, while propidium iodide (PI) stains the DNA of cells with compromised cell membranes. These reagents are commonly used in flow cytometry and fluorescence microscopy applications to analyze cell death and cellular processes.
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Lab products found in correlation
Flow cytometry, by BD (2 mentions)
Rnase a, by Merck Group (2 mentions)
Apc goat anti rabbit igg, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Flow jo v8, by FlowJo (1 mentions)
Binding buffer, by Sungene Biotech (1 mentions)
Facsuite software, by BD (1 mentions)
Binding buffer, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest pro software 5, by BD (1 mentions)
Annexinv fitc pi double staining kit, by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Apoptosis detection kit, by Merck Group (1 mentions)
Facscan, by Beckman Coulter (1 mentions)
Hoechst 33258, by BD (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
One step tunel apoptosis assay kit, by Keygen Biotech (1 mentions)
Hoechst 33258 solution, by Beyotime (1 mentions)
Annexin 5 binding buffer, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
17 protocols using annexin 5 fitc and pi
1
Apoptosis Assay for Cancer Cells
2
Apoptosis Detection by Flow Cytometry
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Cells were grown in 6 cm dishes to 50% confluence in 5% FBS ± 2 μg/ml doxycycline for 72 h. Cells were incubated with the desired concentration of cisplatin for another 72 h. The conditioned medium from each well was collected; cell monolayers were trypsinized, resuspended in the corresponding conditioned medium, centrifuged at 3000 rpm for 3 min at 4 C, washed twice with cold PBS, and resuspended in annexin V-FITC and PI according to the manufacturer's recommendations (Millipore). Flow cytometry was conducted in the University of Virginia (UVA) Flow Cytometry Core Facility with marker combinations as follows: annexin V and PI negative; cells in early apoptosis are annexin V positive and PI negative; cells in late apoptosis are annexin V and PI positive; necrotic/dead cells are annexin V negative and PI positive [23] . Acquisition was performed on a FACS Calibur™ Flow Cytometry. Analysis was performed using Flow Jo v8.5 (Flowjo, LLC-Ashland, OR, USA).
3
Annexin-V-FITC and PI Staining
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Cells were collected, washed by PBS and resuspended by staining buffer. Adjust the final concentration to 1 × 107 cells/mL. Take 100ul of the suspension, incubate with Annexin-V-FITC and PI (Sigma-Aldrich, USA) in dark. 15 mins later, collect cells and wash again with staining buffer. Then, cells were subjected to FACS analysis by flow cytometer (Becton Dickinson, San Jose, CA, USA).
4
Apoptosis Induction in NB4 Cells
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NB4 cells were fixed with pre-cold 70% ethanol overnight at 4°C and washed with PBS. Then, incubated with 50 µl Propidium Iodide (PI) for 15 min at room temperature and cells were analysis in flow cytometer. Each experiment was repeated at least three times.
NB4 cells were treated of various concentrations of lapatinib for 24 h, and cells were harvested by centrifugation at 3000 r for 5 min. After washed three times with pre-cold PBS, cells were re-suspended in Binding Buffer (Sungene Biotech Co., Ltd., Tianjing, China), and stained by Annexin V-FITC and PI (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature and cells were analysis in flow cytometer. Each experiment was repeated at least three times.
NB4 cells were treated of various concentrations of lapatinib for 24 h, and cells were harvested by centrifugation at 3000 r for 5 min. After washed three times with pre-cold PBS, cells were re-suspended in Binding Buffer (Sungene Biotech Co., Ltd., Tianjing, China), and stained by Annexin V-FITC and PI (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature and cells were analysis in flow cytometer. Each experiment was repeated at least three times.
5
Annexin V-FITC/PI Apoptosis Assay
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Annexin v-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining was used to detect apoptosis. According to the protocol, 661W cells were harvested after different treatments, and 100 μL of an Annexin v-FITC and PI (Sigma-Aldrich) mixture was added to the cells. Then flow cytometry was used to detect fluorescent cells. The number of apoptotic cells and the ratio of apoptosis in 661W cells were analyzed using BD FACSuite software.
6
Assessing Cisplatin-Induced Apoptosis in A549/DDP Cells
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Transfected and untransfected A549/DDP cells (3.5×105 cells/ml) were seeded into 12-well plates and incubated with 0, 1.0 or 2.0 µg/ml DDP for 24 h. The A549/DDP cells were then washed with 1X PBS and resuspended in 100 µl binding buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Next, Annexin V-FITC and PI (Sigma-Aldrich; Merck KGaA) were added for 30 min at 37°C in the dark. Following dilution with 400 µl binding buffer, staining was analyzed within 1 h by flow cytometry. The fluorescence intensity (green, FL1-H; red, FL2-H) was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). CellQuest Pro software 5.1 (BD Biosciences) was used for acquisition and analysis of data.
7
Quantification of Cell Death by Photodynamic Therapy
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Cell death after photodynamic effect was quantified for selected condition using the AnnexinV-FITC/PI double staining kit (Sigma Aldrich). The cell death of LLC cells was investigated 4 h after photodynamic treatment. Briefly, the LLC cells (1 · 105 per well) were cultured overnight in 12-well plates. Then cells were subjected to redaporfin-mediated photodynamic effect (described above), collected by centrifugation and washed twice in Hank’s Balanced Salt Solution (HBBS). The cells were then resuspended in 500 μL binding buffer and stained with annexin V-FITC and PI according to the manufacturer’s instructions (Sigma Aldrich). Stained LLC cells were then examined using Guava® easyCyte™ flow cytometer. Obtained data were analyzed using FlowJo 10.5.3 software (BD Bioscience).
8
Apoptosis Quantification in IL-1 Stimulated Cells
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After transfection and IL-1 administration, the HC-A cells (5 × 105) in 6-well plates were examined for apoptosis using the Annexin V-FITC/PI apoptosis detection kit (Invitrogen, Carlsbad, CA). In the presence of 50 g/mL RNase A, 1 × 105 cells from each sample were washed twice with PBS and stained with Annexin V-FITC and PI according to the manufacturer's protocol (Sigma-Aldrich). The FACS can discriminate between apoptotic and non-apoptotic cells (Beckman Coulter, Fullerton, CA). FlowJo software was used to quantify the data (Tree Star Inc. Ashland, OR).
9
Apoptosis Quantification in DLBCL Cells
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Apoptosis was measured using an Apoptosis Detection Kit (Sigma). DLBCL cells (2 × 105 cells per well) were seeded in 12‐well plates. After 24 h of treatment, the cells were collected and treated with Annexin V‐binding buffer, then labelled with Annexin V‐FITC and PI (Sigma). The percentage of apoptotic cells was assessed using flow cytometry.
10
Annexin V-FITC and PI Apoptosis Assay
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We collected NR8383 cells with EDTA-free pancreatin (Gibco) and washed them with PBS. Cells (1 × 106 cells) were then resuspended in binding buffer, and 1 × 105 cells were subsequently stained with Annexin V-FITC and PI (Sigma–Aldrich) for 20 min. Afterward, cells were incubated in the dark for 1 h at room temperature. We used FACScan (Beckman Coulter, Fullerton, CA, USA) to detect the population of apoptotic cells and FlowJo software (Treestar, Ashland, OR, USA) to analyze them.
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