The percentage of apoptotic cells and the cell-cycle distribution of cells was determined using the BD Annexin V-FITC/PI Assay Kit and a Cycle Staining Kit, respectively. Both assays were according to the manufacturer’s instructions. Cell apoptosis and cell-cycle analyses were performed by flow cytometry (Accuri model C6).
Annexin 5 fitc pi assay kit
Manufactured by BD
Sourced in United States
The Annexin V-FITC/PI assay kit is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death, in cell samples. The kit utilizes the binding properties of the protein Annexin V, which has a high affinity for the phospholipid phosphatidylserine, a marker for apoptotic cells. The kit also includes the DNA-binding dye propidium iodide (PI) to distinguish between early apoptotic, late apoptotic, and necrotic cells. This assay provides a standardized method to analyze cellular viability and the stages of apoptosis through flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Accuri model c6, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Anti rabbit and anti mouse hrp conjugated secondary antibodies, by Cell Signaling Technology (1 mentions)
Pgsk3α β, by Cell Signaling Technology (1 mentions)
Hplc grade formic acid, by Fujifilm (1 mentions)
Lithium chloride (licl), by Merck Group (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Methanol, by Merck Group (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Facscan flow cytometry, by BD (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Matrigel, by BD (1 mentions)
Matrigel high concentration, by Corning (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
13 protocols using annexin 5 fitc pi assay kit
1
Cell Apoptosis and Cell Cycle Analysis
2
Apoptosis and Cell Cycle Analysis
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Cells were seeded into 6‐well plates and then collected after 48 hours of transfection with an siRNA or plasmid. The number of apoptotic cells was determined using a BD Annexin V‐FITC/PI Assay kit according to the manufacturer's instructions. For cell cycle analysis, cells were harvested and washed 3 times, and the cell cycle distribution of cells was determined using a Cycle Staining kit (CCS012, Multisciences, Hangzhou, China) according to the manufacturer's instructions. Cell apoptosis and cell cycle analyses were performed by flow cytometry (Accuri model C6).
3
Annexin V-FITC/PI Assay for Apoptosis
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The apoptosis rate was detected using Annexin V-FITC/PI assay kit (BD Bioscience, San Jose, CA, USA). Cells were collected and washed twice with phosphate-buffered saline and dark stained for 15 minutes at room temperature. The apoptosis rate was measured by flow cytometry (FACSCalibur, BD bioscience) and analyzed by Flow Jo software (BD, Ashland, OR, USA).
4
Glioma Cell Apoptosis Induced by 1
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flow cytometry was used to analyze the effect of 1 on the apoptosis of glioma cells LN229. Cells were treated with different concentrations of 1 for 24 h. Then, the cells were collected and washed twice with cold PBS for five minutes (1000 rpm). The cells were analyzed for apoptosis by flow cytometry (BD, Franklin Lakes, NJ, USA) using the Annexin V-FITC/PI assay kit.
5
Measuring Cardiomyocyte Apoptosis
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Cell apoptosis was measured by Annexin V-FITC/PI assay kit (BD, USA). After digestion, CMECs were washed 2 times with cooled PBS. Then, cells were suspended with 1× binding buffer and added with Annexin V-FITC and PI 2.5 μL each. CMECs were incubated under light avoidance conditions at room temperature for 15 minutes. Apoptosis was detected by flow cytometry (Partec, Germany), and data were analyzed by FlowJo software.
6
Apoptosis Monitoring via Flow Cytometry
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Cells were infected with viruses at an indicated MOI, collected, and stained with PI (1 μg/mL) alone or together with Annexin V-FITC (0.1 g/mL) using the Annexin V-FITC/PI assay kit (BD Pharmingen, USA), according to the manufacturer’s instructions. Subsequently, 10,000 cells were analyzed and apoptotic cells were identified using a Cytomics FC 500 flow cytometer (Beckman Coulter Inc., USA) on the FL-1 and FL-3 channels.
7
Optimized Assay Protocols for Cell Studies
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Extra pure grade n-hexane and ethyl acetate were purchased from Duksan Pure Chemical (Korea). HPLC-grade formic acid was purchased from Wako (Japan). HPLC-grade acetonitrile was obtained from Fisher Scientific Korea (Korea). Methanol, 3-(4,5-dimethylthiazol-e-yl)-2,5-diphenyl tetrazolium (MTT), propidium iodide (PI) solution, LiCl, crystal violet solution (1%), 37% formaldehyde, and TRI reagent were obtained from Sigma-Aldrich (USA). A RevertAid First Strand cDNA Synthesis Kit and Power SYBR Green PCR Master Mix were purchased from Thermo Fisher Scientific. An Annexin V-FITC/PI Assay Kit and antibodies against cyclin D1 and E2F-1 were purchased from BD Bioscience (USA). Antibodies against WNT2B, c-Myc, Bcl-2, PCNA, and β-actin were purchased from Santa Cruz Biotechnology (USA). Antibodies targeting GSK3α/β, p-GSK3α/β, β-catenin, PARP, CDK4, and HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Cell Signaling Technology (USA).
8
Cell Proliferation, Migration, and Apoptosis Assays
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Cell Counting Kit-8 (CCK-8; Beyotimes) was used to determine cell proliferation. LN229 and U251 cells were seeded into a 96-well plate at a density of 3,000/well and cultured at 37°C with 5% CO2 for 24 h. Then, per well were interacted with 10 μL CCK-8 solution for another 2 h at 37°C. Finally, the absorbance was measured at 450 nm using a microplate reader (Bio-Rad).
Transwell assays were carried out using chamber inserts precoated with or without Matrigel (BD Biosciences, San Jose, CA, USA) to analyze the migration and invasion of LN229 and U251. Experiments were repeated in triplicate independently. Details of the procedure are based on the literature as described previously [17 (link)].
For apoptosis analysis, LN229 and U251 cells were gathered and Annexin V-FITC/PI assay kit (BD Biosciences) was used according to the manufacturer’s protocol. Then, FACScan® flow cytometry (BD Biosciences) was carried out to detect cell apoptotic rate.
Transwell assays were carried out using chamber inserts precoated with or without Matrigel (BD Biosciences, San Jose, CA, USA) to analyze the migration and invasion of LN229 and U251. Experiments were repeated in triplicate independently. Details of the procedure are based on the literature as described previously [17 (link)].
For apoptosis analysis, LN229 and U251 cells were gathered and Annexin V-FITC/PI assay kit (BD Biosciences) was used according to the manufacturer’s protocol. Then, FACScan® flow cytometry (BD Biosciences) was carried out to detect cell apoptotic rate.
9
Quantifying Rutin-Induced Apoptosis in SiHa Cells
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Rutin-induced apoptosis induction in SiHa cancer cells was quantified using an Annexin V-FITC/PI assay kit (BD Biosciences, Billerica, MA, USA). Briefly, SiHa cancer cells (in 12-well plates) at a density of 5 × 105 cells per mL wer treated with rutin doses (0, 80, 120, and 160 µm) for 48 h. After 48 h of incubation, SiHa cells were washed twice with cold PBS (phosphate-buffered saline) buffer and then the cells were re-suspended in binding buffer (500 μL). Thereafter, cells were stained with annexin V-FITC/PI and left to incubate for 15 min in the dark at room temperature. Lastly, the stained cells were analyzed by using flow cytometry.
10
Investigating Cisplatin's Cytotoxic Effects
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Ham's F12K medium, RPMI‐1640 medium, and phosphate buffer solution (PBS) were purchased from HyClone Laboratories Inc. Trypsin–EDTA (0.25%), fetal bovine serum (FBS), and penicillin–streptomycin solution were purchased from Gibco Laboratories Inc. Cisplatin was purchased from Sigma‐Aldrich Laboratories. The CCK‐8 assay kit was purchased from the Dojindo Chemical Research Institute. The Annexin V‐FITC/PI assay kit was purchased from BD Biosciences. RNeasy Mini SYBR Premix Ex Taq and the PrimeScript 1st strand cDNA Synthesis Kit were purchased from Takara Bio Inc. The reactive oxygen assay kit, GSH/GSSG assay kit, western blot lysis buffer, phenylmethylsulfonyl fluoride (PMSF), western blot transfer buffer, and nitrocellulose membranes were purchased from Shanghai Biotechnology Co Ltd. γ‐GCS and GPx assay kits were purchased from Shanghai Xinle Biotechnology Co Ltd. Bicinchoninic acid protein assay was purchased from Pierce. Primary antibodies GST‐π, MT, multidrug resistance‐1 (MDR‐1), lung resistance protein (LRP), and β‐microtubulin (β‐tubulin), as well as horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse IgG secondary antibodies, were purchased from Abcam. Enhanced chemiluminescence (ECL) chemiluminescence reagents were purchased from Millipore. High concentration matrigel was purchased from Corning.
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