Apoptosis was detected using an Annexin V-FITC/PI double staining kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Cells were seeded at a concentration of 5×103 cells/100 µl/well in 96-well culture plates, then 0.1 µM gefitinib was added 48 h prior to detection. Cells were harvested and washed twice with cold PBS by gentle shaking. Cells were then re-suspended and added to Binding buffer (1X), and cell density was adjusted to 2–5×105/ml. In the dark, 5 µl Annexin V-FITC was added to the cell suspension volume of 195 µl and incubated for 10 min at room temperature prior to the addition of 190 µl binding buffer (1X) and 10 µl propidium iodide (PI). A total of 10,000 events per sample were acquired using a FACS-scan flow cytometer (BD Biosciences, San Jose, CA, USA), and the percentage of cells undergoing apoptosis was analyzed using BD CellQuest™ Pro Software Analysis Tutorial (Version 5.1; BD Biosciences).
Annexin 5 fitc pi double staining kit
Manufactured by BD
Sourced in United States
The Annexin V-FITC/PI double staining kit is a laboratory tool designed to detect and differentiate between apoptotic and necrotic cells. It utilizes the binding properties of Annexin V, a calcium-dependent phospholipid-binding protein, conjugated with the fluorescent dye FITC, and the DNA-binding dye propidium iodide (PI) to provide a comprehensive assessment of cell viability and the mode of cell death.
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Lab products found in correlation
Bcl 2, by Cell Signaling Technology (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Facsscan flow cytometer, by BD (1 mentions)
Mitotracker green probe, by Beyotime (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Transstart green qpcr supermix, by Transgene (1 mentions)
Thiazolyl blue tetrazolium blue mtt, by Merck Group (1 mentions)
Rneasy animal rna isolation kit with spin column, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Sangon (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Flow cytometry, by BD (1 mentions)
Cxp software, by Beckman Coulter (1 mentions)
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
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Mtt assay kit, by Beyotime (1 mentions)
Rt pcr kit, by Yeasen (1 mentions)
Erk1 2, by Affinity Biosciences (1 mentions)
Dapi staining kit, by Beyotime (1 mentions)
Tan 1, by Yuanye Bio-Technology (1 mentions)
18 protocols using annexin 5 fitc pi double staining kit
1
Apoptosis Detection using Annexin V-FITC/PI
2
Annexin V-FITC/PI Apoptosis Assay in Cardiomyocytes
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The Annexin V-FITC/PI double staining kit (BD Biosciences, USA) was used to detect cardiomyocyte apoptosis by flow cytometry. Briefly, cardiomyocytes were collected after IH exposure, washed with ice-cold PBS, and resuspended in 500 μl binding buffer. And then, the cells were incubated with Annexin V-FITC and PI (5 μl each) for 15 min at room temperature while avoiding light. Data were collected with a flow cytometer (BD Biosciences, USA) within one hour and analyzed with the FlowJo software (BD Biosciences, USA).
3
Apoptosis and Mitochondrial Dynamics Evaluation in C2C12 Myoblasts
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C2C12 myoblasts were seeded in 6-well plates and treated with UCF101 or DMSO after differentiation was initiated. The cells were harvested at D1, 3, and 5 and stained according to the manufacturer’s instructions. Apoptosis was evaluated using an Annexin V-FITC PI double staining kit (BD Biosciences, San Diego, CA, USA). The number of mitochondria was measured using a MitoTracker Green probe (Beyotime, Shanghai, China). ROS levels were measured using a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China). Samples were analyzed by flow cytometry (Guava easyCyte, Austin, TX, USA).
4
Dendrobium officinale Cultivation Protocols
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D. officinale (2-year-old) was purchased from the Dendrobium base in Huoshan County, Anhui Province, China (simulated field growing environment). All chemical reagents and solutions were of analytical grade or LC-MS mass spectrometry grade, and methanol, acetonitrile, formic acid, and ammonium bicarbonate were purchased from CNW (Dusseldorf, Germany). Fetal bovine serum (FBS) was purchased from Sangon Biotech (Shanghai, China). RPMI Medium Modified 1640 medium and phosphate-buffered saline (PBS) were purchased from HyClone, Thermo Scientific (MA). Thiazolyl Blue Tetrazolium Blue (MTT) was purchased from Sigma (St. Louis, MO, USA). Annexin V-FITC/PI Double Staining Kit was purchased from BD (Franklin Lakes, NJ, USA). RNeasy™ Animal RNA Isolation Kit with Spin Column was purchased from Beyotime (Shanghai, China). TransStart® Green qPCR SuperMix and TransScript® Reverse Transcriptase [M-MLV,R-NaseH-] were purchased from TransGen Biotech (Beijing, China).
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D. officinale (2-year-old) was purchased from the Dendrobium base in Huoshan County, Anhui Province, China (simulated field growing environment). All chemical reagents and solutions were of analytical grade or LC-MS mass spectrometry grade, and methanol, acetonitrile, formic acid, and ammonium bicarbonate were purchased from CNW (Dusseldorf, Germany). Fetal bovine serum (FBS) was purchased from Sangon Biotech (Shanghai, China). RPMI Medium Modified 1640 medium and phosphate-buffered saline (PBS) were purchased from HyClone, Thermo Scientific (MA). Thiazolyl Blue Tetrazolium Blue (MTT) was purchased from Sigma (St. Louis, MO, USA). Annexin V-FITC/PI Double Staining Kit was purchased from BD (Franklin Lakes, NJ, USA). RNeasy™ Animal RNA Isolation Kit with Spin Column was purchased from Beyotime (Shanghai, China). TransStart® Green qPCR SuperMix and TransScript® Reverse Transcriptase [M-MLV,R-NaseH-] were purchased from TransGen Biotech (Beijing, China).
5
Quantifying Apoptosis by Flow Cytometry
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The quantification of apoptosis was determined by flow cytometry using an annexin V-FITC/PI double staining kit (BD Pharmingen, San Diego, CA, USA), according to the manufacturer’s instruction. Briefly, cells were treated with the desired concentrations of coptisine, for 24 h, in the presence or absence of Z-VAD-FMK (Sigma-Aldrich Chemical Co.). The collected cells were re-suspended in binding buffer, and then stained with PI solution and FITC-conjugated annexin V, for 15 min, in dark, as previously described [47 (link)]. The fluorescence intensities were detected by flow cytometry (BD Biosciences, San Jose, CA, USA) at the Core-Facility Center for Tissue Regeneration, Dong-eui University (Busan, Repbulic of Korea).
6
Quantification of Apoptosis and Necrosis by Flow Cytometry
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After being treated with/without RV, ARV, ORV and PD, approximately 1×106 cells were suspended in 100 μL of PBS, and then 200 μL of 70% ethanol were added and vortex for flow cytometry analysis. Following the treatment, the cells were harvested and washed twice with PBS. Apoptotic or necrotic cells were quantified using an annexin V-FITC-PI double staining kit (BD Biosciences, San Jose, CA, USA). Cells were centrifuged and resuspended in a binding buffer containing FITC-conjugated annexin V and PI. Then, samples were incubated for 15 mins in the dark prior to be assessed by dual-color flow cytometry by FC500 flow cytometer with CXP software (Beckman Coulter, Fullerton, CA, USA). Cells were classified as follows: live cells (Annexin/PI), necrotic cells (Annexin/PI+), early apoptotic cells (Annexin+/PI) or late apoptotic cells (Annexin+/PI+). The data were analyzed by flow cytometry CXP analysis software. To establish the confidential conclusions, the experiments were repeated at least three times.
7
Apoptosis Analysis in Transfected OS Cells
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Apoptosis analysis was conducted using an AnnexinV-FITC/PI double staining kit (BD Biosciences). Briefly, the transfected OS cells were harvested and subjected to resuspension in 1 × Binding Buffer and then treated with Annexin V-FITC/PI according to the kit instructions. After incubating for 15 min, a flow cytometer (BD Biosciences) was employed to detect the Apoptosis of the cells.
8
Apoptosis Analysis of HepG2 Cells
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HepG2 cells were seeded in 6-well plate at a density of 1 × 106 cells per well and treated with PBS (control), TP (50 nM), arctiin (50 μM), and a combination of pretreatment with arctiin (50 μM) for 12 h followed by TP (50 nM) treatment in order. After 24 h, the cells were washed with PBS twice, and 0.25% trypsin was used to detach the cells. Then, cells were centrifuged, and the sediment collected was used to determine the apoptosis rate with the aid of flow cytometry (BD Biosciences, CA, USA) using Annexin V-FITC/PI double staining kit.
9
Annexin V-FITC/PI Apoptosis Assay
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Cells were treated with increasing concentrations of Evo for 2 days. Adherent and floating cells were collected and washed with PBS. Cells were stained with Annexin V-FITC and PI using the commercially available Annexin V-FITC/PI double staining kit (BD Bioscience). Fluorescence intensity was analyzed by flow cytometry using a FACSCalibur® flow cytometer (BD Biosciences) and analyzed using Flowing software (Cell Imaging and Cytometry (CIC) Core, Turku Bioscience) 31 (link).
10
Apoptosis and Senescence Detection in Vascular Endothelial Cells
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Cell apoptosis was detected by flow cytometry. Cells treated or transfected with different agents for 48 h were harvested and fixed. Cell apoptosis was detected by using an Annexin V-FITC/PI Double Staining Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. The percentages of apoptotic VECs were analyzed using a BD FACS Calibur flow cytometer (BD Biosciences). Aged cells were detected by using a β-Galactosidase staining kit (Jiancheng Biological Company, Nanjing, China) according to the manufacturer's recommended protocol. Senescence-associated β-galactosidase (SA-β-Gal) in aged cells was detected as previously described [25 (link)]. Cellular images were captured using a microscope (Motic, Xiamen, China). Each experiment was repeated 3 times.
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