Cells were seeded and incubated overnight at a density of 1 × 105 cells/ml in 24-well plates. After incubation for 24 h with increasing concentrations of AELE, samples were collected, centrifuged at 1500 rpm and 4 °C, resuspended in suspension buffer and stained with Annexin and Propidium Iodine (PI) (Annexin V–fluorescein isothiocyanate [FITC] Apoptosis Detection Kit, Abcam). Samples were immediately analyzed using Accuri C6 flow cytometer.
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit is a laboratory reagent used to detect and quantify the apoptotic cells in a given sample. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the cell surface during apoptosis. The kit utilizes FITC-conjugated Annexin V to label the apoptotic cells, which can then be detected and analyzed using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Acea novocyte flow cytometer, by Agilent Technologies (2 mentions)
Acea novoexpress software, by Agilent Technologies (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Facscan flow cytometer, by Beckman Coulter (1 mentions)
Facsaria, by BD (1 mentions)
Diva 6, by BD (1 mentions)
Annexin 5 fitc, by Abcam (1 mentions)
Facscan, by Beckman Coulter (1 mentions)
Annexin 5 fitc, by Solarbio (1 mentions)
Fluorescence spectrophotometer, by Hitachi (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Isopropyl alcohol, by BD (1 mentions)
Facscan, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Antibodies against icam 1, by Abcam (1 mentions)
Flow cytometry, by BD (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
35 protocols using annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit
1
Assessing AELE-Induced Apoptosis
2
U937 Cell Apoptosis Assay
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Six well plates were used to seed U937 cells at a density of 1 × 105 cells/well for 4 h before treatment with the following concentrations of UD extract: 0.4, 0.8, 1.6, and 2.4%. Untreated cells were used as controls. Cells were collected and centrifuged after 48 and 72 h of treatment, and the pellets were stained with both annexin V and PI (annexin V–fluorescein isothiocyanate (FITC) Apoptosis Detection Kit, Abcam, Cambridge, UK) and immediately analyzed using the Accuri C6 flow cytometer, as previously described [26 (link),27 (link)]. Upon apoptosis induction, phosphatidyl serine is translocated to the outer leaflet of the cell membrane, and living cells exclude PI from the cytoplasm. Therefore, living cells will stain negative for both FITC–annexin V and PI, whereas those that stain positive for both can be classified as apoptotic dead cells [28 (link)].
3
Annexin V-FITC Apoptosis Assay
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After 48 h of transfection, an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Abcam, Cambridge, UK) was used to detect cell apoptosis, according to the manufacturer's protocol. Following harvesting and washing twice with PBS, the cells were stained with Annexin V-FITC and propidium iodide (PI), and incubated for 15 min at room temperature in the dark. Cell apoptosis was subsequently detected using a FACScan flow cytometer (Beckman Coulter, Inc., Brea, CA, USA).
4
Annexin V Apoptosis Detection Assay
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Early in apoptosis, phosphatidylserine (PTS) is translocated to the outer cell membrane and can be identified by binding of Annexin V, a ligand for PTS. Apoptotic Capan-1 and SW1990 cells were quantified using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (abcam, UK) after cells were treated with APY606 at different concentrations (6.25 and 12.5 μg/mL) for 24 h. Briefly, cells were trypsinized and washed twice with cold PBS, and then the cells were resuspended at a density of 1×106 cells/mL in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Thereafter, cells were incubated with 5 μL annexin V-FITC and 5 μL PI in the dark for 15 min at room temperature and subjected to flow cytometric analysis (FACSAria, BD Biosciences). In total, 10,000 events were analyzed in each sample. Data analysis was performed with Diva 6.0 software (BD Biosciences).
5
Annexin V-FITC Apoptosis Assay in MDA-MB-231 Cells
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MDA-MB-231 cells were cultured and treated as described in the previous sections. After incubation for 24 h with AELE (261 and 522 μg/mL) and topotecan (20 μM), samples were collected, centrifuged at 1500 rpm and 4 °C, resuspended in suspension buffer, and then stained with Annexin V-FITC (Annexin V–fluorescein isothiocyanate [FITC] Apoptosis Detection Kit, Abcam) according to the manufacturer’s instructions. Samples were then analyzed using the Guava easyCyte™ flow cytometer [40 ].
6
Detecting Cellular Apoptosis via Annexin V-FITC
7
Apoptosis and Necrosis Quantification
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Apoptotic and necrotic cell populations were determined using Annexin V- Fluorescein isothiocyanate (FITC) apoptosis detection kit (Abcam Inc., Cambridge Science Park Cambridge, UK) coupled with two fluorescent channels flow cytometry. After treatment with Er2O3-NPs for 72 h and doxorubicin as a positive control, Hep-G2 cells were collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Harvested cells are incubated in dark with Annexin V-FITC/ propidium iodide (PI) solution for 30 min at room temperature, then injected via ACEA Novocyte flowcytometer (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed for FITC and PI fluorescent signals using FL1 and FL2 signal detector, respectively (λex/em 488/530 nm for FITC and λex/em 535/617 nm for PI). For each sample, 12,000 events were acquired and positive FITC and/or PI cells are quantified by quadrant analysis and calculated using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA).
8
Quantitative Evaluation of Apoptosis
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Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit was purchased from Abcam Inc. (Cambridge, UK) and used for quantitative evaluation of apoptosis-induced cells upon treatment with phloroglucinol and/or H2O2. After treatment, collected cells were suspended in annexin binding buffer containing annexin V- FITC and propidium Iodide (PI) following the manufacturer’s instructions. The fluorescence of 10,000 events was then acquired using a flow cytometer (Becton Dickinson, San Jose, CA, USA). Annexin V-positive cells were considered as apoptosis-induced cells as described previously [27 (link)].
9
Apoptosis Detection by Annexin V-PI Staining
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Cells were plated in six-well plates at a density of 0.5×105 cells/well and incubated for 24 h. After treatment, Annexin V/PI staining was performed to detect and analyze apoptosis using flow cytometry. Cells were collected and centrifuged at 1500 rpm at 4°C for 5 min. The pellet was suspended in 500 µL suspension buffer, in addition to 5 µL Annexin and 5 µL PI (Annexin V–fluorescein isothiocyanate [FITC] Apoptosis Detection Kit, Abcam, Cambridge, UK) and immediately analyzed by the flow cytometer. Annexin V is a Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is translocated from the cytoplasmic surface to the outer leaflet of the cell membrane upon apoptosis. The cell membrane is impermeable to PI and hence PI is excluded from living cells. Cells that are stained negative for FITC–Annexin V and negative for PI are considered living cells. Cells that are stained positive for both FITC-Annexin V and PI are either at the end stage of apoptosis, undergoing necrosis, or are already dead.
10
Apoptosis Detection by Annexin V-FITC and PI
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This technique was employed to determine the type of cell death induced by co-treatments. Annexin V detects the translocation of phosphatidylserine from the inner leaflet to the surface of the plasma membrane, which is characteristic of apoptotic cells, whereas propidium iodide (PI) detects late apoptotic and ruptured cells with permeabilized plasma membrane. Cultured cells were seeded in six-well plates and simultaneously transfected with siRNA or expression vector targeting RBBP6 for 24 hours and treated for additional 24 hours with apoptosis-inducing agents (100 µM GABA and 0.25 µM camptothecin). The treated cells were then trypsinized, transferred to 15 mL tubes, pelleted for 2 minutes at 1,500 rpm, and resuspended in 100 µL of 1X binding buffer (annexin V-fluorescein isothiocyanate [FITC] Apoptosis Detection Kit; Abcam, Cambridge, UK) at a concentration of 1×104 cells/mL. The cell solution was then transferred to 1 mL tubes, and 5 µL of annexin V-FITC and 5 µL of PI were added. This was followed by gentle vortexing and incubation for 15 minutes at room temperature in the dark. Four hundred microliters of 1X binding buffer was then added to each tube, and the cell solutions were analyzed by flow cytometry within 1 hour.
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