The apoptosis of cancer cells was determined by Annexin V‐FITC detection kit (C1062S) (Beyotime Biotech). Briefly, A549 and Capan‐2 cells were cultured overnight in a sixwell plate at a density of 2 × 105 cells per well and treated in varying concentrations of drugs for 48 h. The final concentrations of TmSm and YM155 were kept at 2 and 0.15 μM, respectively. The harvested cells were resuspended in 200 μl of binding buffer and stained with Annexin V‐FITC and propidium iodide (PI) at 25°C. The stained cells were analyzed using FCM equipped for FITC and PI.
Annexin 5 fitc detection kit
Manufactured by Beyotime
Sourced in China
The Annexin V-FITC detection kit is a laboratory reagent used to identify and quantify apoptotic cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the outer membrane of apoptotic cells. The FITC (fluorescein isothiocyanate) label allows for the detection of Annexin V-positive cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Accuri c6, by BD (6 mentions)
Flow cytometry, by BD (2 mentions)
Pxd101, by Selleck Chemicals (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Ab133327, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab234436, by Abcam (1 mentions)
Ab2324, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab32053, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Cck 8 detection kit, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Cleaved parp1, by Abcam (1 mentions)
Goat anti rabbit igg secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
29 protocols using annexin 5 fitc detection kit
1
Apoptosis Analysis of Cancer Cells
2
Evaluating Ovarian Cancer Cell Apoptosis
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To test whether USP5 amplification affects the proapoptosis activity of PXD101, primary ovarian cancer cells were isolated from 8 patients who were admitted to Shanghai General Hospital as previously described [41 (link)] after written informed consent was obtained. Genomic DNA was isolated from these cells and CNV was detected as described above. These cells were seeded into 6-well plates, cultured overnight and exposed to PXD101 (1 μM; Selleck Chemicals, Houston, TX, USA), cisplatin (5 μg/ml; Aladdin, Shanghai, China) or vehicle (DMSO). After 48 h, cell apoptosis was assessed by Annexin V-FITC detection Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol.
3
Quantitative Apoptosis Assay by Flow Cytometry
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Quantitation of apoptotic cells was performed using the Annexin V-FITC detection kit (Beyotime) according to the manufacturer’s protocol. Briefly, cells were cultured on a 6-well dish at the density of 1× 106 cells/well. Following treatment, cells were collected, washed with PBS and re-suspended in 195 μL binding buffer containing 5 μL Annexin V-FITC and 10 μL PI, followed by incubation for 15 min at room temperature in the dark. Finally, fluorescence was quantified by flow cytometry, where apoptotic cells were defined as positive staining for Annexin V or PI.
4
Quantifying Apoptosis in Drug-Treated Cells
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Quantification of drug treated cells was performed using the Annexin V-FITC detection kit (Beyotime, Shanghai, China) according to the instruction manual and the previously published protocol (Yan et al., 2009 (link)). After treatment with ActD, BMH21 and CX5461 for 24 h, the cells were collected and washed once with pre-chilled PBS. The cells were gently resuspended in the binding solution, followed by the addition of 5 μl Annexin VFITC and 10 μl propidium iodide (PI) dye liquor. The cell suspension was then mixed gently and incubated for 20 min at room temperature in the dark. The cells were re-suspended 2–3 times during the incubation to improve staining. After the completion of the dyeing, the sample was filtered through a 400-mesh screen to perform the FCM test. FITC Annexin-V staining was detected in the FL1 channel, whereas PI staining was monitored in the FL2 channel. Data were analyzed with Summit software.
5
Annexin V-FITC Apoptosis Assay in U87 Glioma Cells
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The U87 glioma cells were collected and then suspended in 500 μL of 1X binding buffer. Then, Annexin V-FITC (5 μL) was added to the suspension followed by PI (10 μL) (Annexin V-FITC detection Kit, Beyotime Biotechnology). The cells were mixed evenly and refrigerated at 4°C for 10 min in the dark. Flow cytometer (Accuri C6, Becton-Dickinson, US) was used to detect the apoptotic cells.
6
Annexin V-FITC Apoptosis Assay
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After the harvested cells were washed twice with PBS, they were resuspended in binding buffer (500 μL) containing 5 μL of Annexin V-fluorescein isothiocyanate (FITC) (Annexin V-FITC Detection Kit, Beyotime Biotechnology, Shanghai, China). Then, 10 μL of PI was added to the suspension and the cells were incubated for a further 10 min period. The degree of apoptosis was determined using a FACSCalibur cell analyser (Accuri C6, Becton-Dickinson, US).
7
Annexin V-FITC Assay for Apoptosis and Necrosis
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Annexin V-FITC detection kit (Beyotime, Shanghai, China) was used to detect apoptosis and necrosis in R28 cells after glutamate treatment. After incubation with glutamate for different time points, R28 cells were collected using EDTA-free trypsin and then resuspended with PBS. After that, cells were stained with 5 μL of Annexin V-FITC and 10 μL of PI for 15 min at room temperature in the dark according to the manufacturer’s instructions. Next, the stained cells were analyzed by flow cytometry. The percentage of Annexin V+ cells indicated cell death rate, which was consisted of Annexin V+/PI− (early apoptosis) cells and Annexin V+/PI+ (necrosis and late apoptosis) cells while both Annexin V and PI negative cells were considered as living cells. For each sample, 3 × 104 cells were collected.
8
Annexin V-FITC Apoptosis Detection
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The apoptosis was detected using a commercial annexin V-FITC detection kit (Beyotime Biotechnology, Shanghai, China). After treatment with indicated concentrations of DHT, the cells were collected, washed twice with PBS, and resuspended with 100 μL of binding buffer. The cell suspension was incubated for 10 min at room temperature with 5 μL of annexin V-FITC and 10 μL of propidium iodide (PI). The percentage of apoptotic cells was detected by flow cytometry (Beckman CytoFlex, Brea 92822, CA, USA).
9
Hirsuteine Modulates Apoptosis Pathways
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Hirsuteine (ST17300105; 5 mg/dose; purity ≥98%) was acquired from Shanghai Standard Technology Co., Ltd. The CCK-8 Detection kit, BSA, and Annexin V-FITC Detection kit were acquired from Beyotime Institute of Biotechnology. B-cell lymphoma-2 (Bcl-2; ab32124; dilution, 1:1,000), Bcl-2-associated X protein (Bax; ab32503; dilution, 1:1,000), Cyclin B1 (ab32053; dilution, 1:1,000), CDK1 (ab133327; dilution, 1:1,000), Apaf1 (ab234436; dilution, 1:1,000), cytochrome C (ab133504; dilution, 1:1,000), cleaved-caspase3 (ab32042; dilution, 1:1,000), cleaved-caspase9 (ab2324; dilution, 1:1,000), cleaved-PARP1 (ab32064; dilution, 1:1,000), β-actin (ab8227; dilution, 1:5,000) antibodies, goat anti-rabbit IgG secondary antibody (ab6721; dilution, 1:20,000) were purchased from Abcam. Other reagents were analytical reagent grade and from commercial sources.
10
Calpain-2 Mediated ER Stress Apoptosis
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Special reagents included rat non-tumor BRL-3A cells (Number: KCB92013YJ, Kunming Cell Bank of Chinese Academy of Sciences, China), fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, New York, NY, United States), Acrylamide, bisAcrylamide, ammonium peroxydisulfate, glycine, Tri-hydroxymethyl aminomethane, 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2-H- tetrazolium bromide (MTT), Tween 20, DTT, dimethyl sulfoxide (Genview, League City, TX, United States), N-succinyl-Leu-Leu-Val-Tyr-AMC, antibodies against GRP78, PERK, ATF4, CHOP, and caspase-12 (Abcam, Cambridge, United Kingdom), antibodies against p-PERK (Affinity Biosciences, Cincinnati, OH, United States), calpain-2 and β-actin (Cell Signaling Technologies, Danvers, MA, United States), caspase-3 and secondary antibodies (Boster Biological Engineering, Wuhan, China), polyvinylidene difluoride membranes and enhanced chemiluminescence kit (Millipore, Burlington, MA, United States). Calpain-2-specific and control small interfering RNAs (siRNAs) and siRNA dilution buffer were produced by Santa Cruz Biotechnology (Santa Cruz, CA, United States), and the Annexin V-FITC Detection Kit, ER-tracker Red, and Fluo-3 AM were obtained from Beyotime Institute of Biotechnology (Nanjing, China).
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