Apoptotic cells were detected using an Annexin V/PI-FITC kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Briefly, cells were seeded in a 6-well plate (1×106 cells/well) and incubated with 10 ng/ml OSM, 1 µM Sal, or both (10 ng/ml OSM + 1 µM Sal) for 6 days at 37°C and 5% CO2, with 1 µg/ml 5-FU and culture medium used as positive and negative controls, respectively. After washing with PBS, cells (5×105) were resuspended in binding buffer (500 µl) and stained with Annexin V-FITC (5 µl) and PI (5 µl) in the dark at room temperature for 5 min before analysis on a flow cytometer within 1 h. The experiment was repeated three times.
Annexin 5 fitc pi kit
Manufactured by Keygen Biotech
Sourced in China
The Annexin V-FITC/PI kit is a laboratory tool used to detect and quantify apoptosis in cell samples. It combines Annexin V, a protein that binds to phosphatidylserine, and propidium iodide, a DNA-binding dye, to differentiate between viable, early apoptotic, and late apoptotic/necrotic cells through flow cytometry analysis.
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Lab products found in correlation
Facscalibur, by BD (9 mentions)
Flow cytometry, by BD (3 mentions)
Cell cycle detection kit, by Keygen Biotech (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Facscalibur system, by BD (2 mentions)
Epics xl flow cytometer, by BD (2 mentions)
Facscan, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Fc500 flow cytometer, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Ferric ammonium citrate, by Merck Group (1 mentions)
Deaprotinin, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions)
4 6 diamidino 2 phenylindol dapi, by Merck Group (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Alpha modified eagle s medium a mem, by Thermo Fisher Scientific (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
53 protocols using annexin 5 fitc pi kit
1
Apoptosis Detection by Annexin V/PI Assay
2
Annexin V/PI-FITC Flow Cytometry Assay for Hypoxia-Induced Apoptosis
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Flow cytometry was performed to analyze cell apoptosis in hypoxia using an Annexin V/PI-FITC kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. In brief, the cells were seeded in a 6-well plate (1x106 cells/well) and cultured under hypoxic conditions. The cells were then treated with ATO (5, 10 or 15 µM) for 24 h, with 50 µM DDP and DMSO used as a positive and negative control, respectively. After washing with PBS, the cells (5x105) were resuspended in binding buffer (500 µl) and stained with Annexin V-FITC (5 µl) and PI (5 µl) in the dark at room temperature for 5 min. The cells were washed with PBS and analyzed within 1 h using a Beckman Coulter FC500 Flow Cytometer with the CellQuest Pro software (version 6.0; BD Biosciences). The experiment was performed for a total of three times.
3
Apoptosis Measurement by Annexin V-FITC/PI
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Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining was used to detect the apoptotic rates of the three groups of cells. The Annexin V-FITC/PI kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Cells (106) cells in the logarithmic growth were collected for the analysis of apoptosis, which was performed according to the kit instructions. The apoptotic rates were measured using a flow cytometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A compensation adjustment of fluorescence was performed prior to the assessment.
4
Apoptosis Assay of T24 Cells
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T24 cells were cultured in RPMI-1640 medium with or without GEM (0.15 μM or 0.75 μM). After transfection for 48 h, cells were harvested and stained with an Annexin V-FITC/PI kit (KeyGEN Biotech, Nanjing, China). Propidium iodide (PI) was used to discriminate between apoptotic cells with membrane integrity (Annexin V+/PI–) and necrotic cells that lost membrane integrity (Annexin V+/PI+).
5
Apoptosis Quantification by Flow Cytometry
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Annexin V-FITC/PI kit (KeyGEN Biotech, Nanjing, China) was used to detect apoptotic cells by flow cytometry. Briefly, HCT116-Control, HCT116-ZNF575, RKO-Control, and RKO-ZNF575 cells were collected, washed and incubated with cold PBS for 1 h, and resuspended in 500 μl binding buffer supplied by the kit. Next, the cells were incubated with 5 μl Annexin V-FITC for 15 min at room temperature, followed by incubation with 5 μl PI incubation for 5 min at room temperature. After washing with PBS for three times, the cells were loaded and analyzed for NoVo expression.
6
Apoptosis Analysis by Flow Cytometry
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After the cell intervention, the cells were collected and labeled with Annexin V-FITC/PI kit (NanJing KeyGen Biotech Co., Ltd., China) and then detected by flow cytometry for apoptosis.
7
Annexin V-FITC/PI Cell Staining
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WJ MSCs were treated by using Annexin V-FITC/PI kit (KeyGEN bioTECH, Nanjing, China) according to the manufacturer's instruction. The stained cells were detected with the BD FACSCalibur [19 (link)].
8
Apoptosis Evaluation by Annexin-V/PI Assay
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Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining were performed using an Annexin-V-FITC/PI kit (Keygen, Biotech, Nanjing, China) according to the manufacturer’s instructions. Briefly, DU145 and PC-3 cells were cultured in 6-well plates and treated with different doses of SAHA for 48 h. Cells were then harvested using 0.05% trypsin and washed twice with cold PBS, and then resuspended in binding buffer. Then, 1×105 cells in 100 μl binding buffer were added to a 5 ml tube, and 5 μl of Annexin-V-FITC reagent and 5 μl of PI were added to each tube. Cells were gently mixed and incubated for 15 min at room temperature. 400 μl binding buffer was then add to each tube. The samples were analyzed by flow cytometry (BD Biosciences). All experiments were performed in triplicate.
9
Apoptosis Quantification by Flow Cytometry
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Cell apoptosis was assessed by flow cytometry using Annexin V-FITC/PI kit (Nanjing KeyGen Biotech. Inc., Nanjing, China) according to the manufacturer's protocol. Briefly, after administration, H9C2 cells were collected with ethylenediaminetetraacetic acid-free trypsin and washed with PBS twice. Then, the cells were resuspended in 200 µL of binding buffer, mixed with 2 µL of annexin V-FITC (fluorescein isothiocyanate) and 2 µL of propidium iodide (PI), and incubated at room temperature for 10 min in the dark. The apoptotic rate of the cells was analyzed using an Attune ®NxT Acoustic focused flow cytometer (Thermo Fisher Scientific, Massachusetts, USA). The percentage of apoptotic cells was calculated after treatment using computer software. Apoptotic rate = number of apoptotic cells / (number of apoptotic cells + normal cells) × 100%.
10
Cellular Apoptosis Pathway Analysis
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N-acetyl-cysteine (NAC), Ferric ammonium citrate (FAC), 2′, 7′-dichlorodihy-drofluorescein diacetate (H2DCF-DA), calcein-AM, Hoechst33258, 4′, 6-diamidino-2-phenylindol (DAPI), penicillin, streptomycin, leupeptin, pepstatin A, deaprotinin, phenylmethylsulfonylfluoride (PMSF), and 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against cytochrome c, bcl-2, bax, cleaved Caspase-3, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and beta-actin (β-actin) were purchased from Abcam (Cambridge, UK). AnnexinV–FITC/PI kit was obtained from KeyGen Biotech (Nanjing, China). Fetal bovine serum and alpha-modified Eagle’s medium (a-MEM) were purchased from Gibco (Waltham, MA, USA). Cell Mitochondria Isolation Kit, Enhanced Chemiluminescence detection kit, Western and immunoprecipitation (IP) Cell Lysis Kit, Bicinchoninic Acid Protein (BCA) Protein Assay Kit, and 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolcarbocyanineiodide (JC-1) were purchased from Beyotime (China). Cell Counting Kit-8 (CCK-8) assay kit was purchased from DojinDo (Japan).
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