Apoptosis was detected with flow cytometry using Annexin V/PI kit (eBioscience; NJ, NY, USA). For this purpose, the cells were incubated with nanoplatforms (4 μg/mL) and nanoplatforms/miR-101 for 90 min in six-well plates. In parallel, the cells incubated with nanoplatforms in the presence or absence of miR-101 were also exposed under laser irradiation (808 nm laser, 1.2 W/cm2) for 10 min. After removing the supernatant, the fresh medium and FBS were added to each well and kept for 24, 48, and 72 h. Finally, the cells were detached from the plate and dyed with Annexin V-FITC/propidium iodide (PI) (based kit protocol, eBioscience) in a dark room. The rates of apoptosis and necrosis were quantified by flow cytometry (FITC detected at 515–545 nm and PI measured at 564–606 nm).
Annexin 5 fitc propidium iodide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Annexin V FITC/propidium iodide (PI) is a laboratory reagent used for the detection and analysis of apoptosis, a type of programmed cell death. 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. Propidium iodide (PI) is a fluorescent dye that binds to DNA and is used to identify necrotic or late-stage apoptotic cells. Together, Annexin V FITC and propidium iodide are commonly used in flow cytometry or fluorescence microscopy to differentiate between viable, early apoptotic, late apoptotic, and necrotic cell populations.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (2 mentions)
Annexin 5 pi kit, by Thermo Fisher Scientific (1 mentions)
Flow cytometry staining buffer, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc pi, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Fli 06, by Merck Group (1 mentions)
Xav939, by Bio-Techne (1 mentions)
Acrylamide bisacrylamide 30 29 1, by Bio-Rad (1 mentions)
Luminata western hrp chemiluminescence substrate, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Facs flow cytometer, by BD (1 mentions)
Facscalibur system facscan, by BD (1 mentions)
13 protocols using annexin 5 fitc propidium iodide
1
Apoptosis Detection by Flow Cytometry
2
Evaluating Cell Viability by Flow Cytometry
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Cell viability was evaluated using flow cytometry according to standard protocols. In total, 10 4 cells were treated with the miR-374a mimic or control oligonucleotides and seeded in 96-well plates, and CDDP (1 μmol/L, Sigma) was added for 2-4 days. Cells were harvested and washed with PBS 48 h after transfection. Cells (~0.5 × 10 6 ) were resuspended in the flow cytometry staining buffer (eBioscience, Frankfurt, Germany) and stained with annexin V-FITC/propidium iodide (PI) (eBioscience) in the dark for 30 min. Cell apoptosis was detected by flow cytometry according to the annexin V-FITC/PI kit protocol and detected using CellQuest Pro software (BD Biosciences, San Jose, CA, US). The flow cytometry data were analyzed using FlowJo 6.0 (BD Biosciences).
3
Cytotoxicity and Apoptosis Assays
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DMBA, TCDD, and alpha-naphthoflavone (α-NF) were purchased from Toronto Laboratories Research (Toronto, Canada). Dulbecco’s Modified Eagle’s Medium (DMEM), TRIzol reagent, Annexin V-FITC/Propidium Iodide (PI) were purchased from Invitrogen Co. (Grand Island, NY). FLI 06, LY294002, sodium azide, and puromycin were purchased from Sigma-Aldrich Co., (St. Louis, MO), whereas XAV-939 was obtained from Tocris Bioscience (Bristol, UK). High Capacity cDNA Reverse Transcription kit and SYBR® Green PCR Master Mix were purchased from Applied Biosystems® (Foster city, CA). DNA primers were obtained from Integrated DNA Technologies (Coralville, IA). Aldefluor® kit was purchased from Stem Cell Technologies (Vancouver, CANADA). Acrylamide/bisacrylamide 30% (29:1) and nitrocellulose membrane were purchased from Bio-Rad Laboratories (Hercules, CA). Vybrant apoptosis assay kit Molecular Probes® were obtained from Thermo Fisher Scientific (Waltham, MA). Primary and Horse Radish Peroxidase (HRP)-conjugated secondary antibodies against target proteins, AhR and CYP1A1 shRNA, and transfection reagent systems were ordered from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Western blot detection kits, Luminata® Western HRP Chemiluminescence Substrates were purchased from EMD Millipore (Billerica, MA).
4
Apoptosis Quantification in GSCs
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After GSCs grown on different concentrations of FNs were treated with carmustine. A dead cell apoptosis kit (annexin V–FITC/propidium iodide (PI), Invitrogen, Carlsbad, CA, USA) was used to assay for apoptosis, according to the manufacturer’s instructions. Collected cells were washed with PBS and resuspended in 100 μL of 1× annexin-V binding buffer to 1 × 106 cells/well. Annexin V–FITC (10 μL) and PI (2 μL) were added to each tube, and cells incubated in the dark for 15 min at room temperature. Analyses were performed using a BD FACS flow cytometer. Cells containing annexin V+/PI− were defined as an early apoptotic population.
5
Apoptosis Quantification in HaCaT Cells
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LPS-induced HaCaT cell apoptosis was analyzed by Annexin V-FITC/propidium iodide (PI) (Invitrogen, Carlsbad, CA, USA) staining. Appropriately treated HaCaT cells were harvested and washed in cold PBS, centrifuged, and re-suspended in annexin-binding solution to which a working solution of FITC-annexin V and PI was added. After 15 min incubation at 25 °C, the samples were immediately analyzed under single laser emitting excitation by flow cytometry.
6
Apoptosis during Cell Differentiation
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Apoptosis was analyzed at 7, 14, and 21 days of differentiation with annexin V FITC/propidium iodide (PI) staining (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Apoptosis was expressed as a percentage of positive cells (annexin V+/PI− and annexin V+/PI+).
7
Annexin V Apoptosis Assay in miR-222 Transfected Cells
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NP cells (5×105 cells/well) were seeded into 6-well plates overnight. miR-222 mimics, or mimics control were added into each well for 72 h. The cells were collected, washed 3 times with cold PBS and resuspended in 1X binding buffer. The cells were stained with dual-staining Annexin V-FITC-propidium iodide (PI; Thermo Fisher Scientific, Inc.) for 20 min at room temperature according to the manufacturer's protocol. The results were analyzed using a FACS Calibur flow cytometer (BD Biosciences) on a BD FACSCalibur system (FACScan, BD Biosciences).
8
Annexin V/PI Cell Apoptosis Assay
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After incubation, cells were harvested with the gentle dissociating buffer TrypLE®, pelleted by centrifugation, washed with PBS, and resuspended in PBS. Cell suspensions were stained with Annexin V/FITC + propidium iodide (PI) according to the manufacturer's instructions (Thermo Fisher Scientifics, #88–8005). After 15 min of incubation in the dark on ice, cells were centrifuged at 2000 rpm for 10 min, resuspended in PBS, and analysed using a FACSCanto II (BD Biosciences). Annexin V/PI scatter plots divided in quadrants were utilised to assess early apoptotic (Annexin V+/PI-), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V-/PI+) cells.
9
Mepivacaine-Induced Apoptosis in SH-SY5Y Cells
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Cell apoptosis assay was performed as described previously [12 (link)]. Briefly, SH-SY5Y cells were exposed to with or without 10 mmol/L mepivacaine. After that, the cells were collected and washed three times with cold PBS, then resuspended in pre-cooled PBS at 4°C. Apoptotic cells were stained with dual-staining Annexin V-FITC-propidium iodide (PI) (catalog number 88–8006-72, Thermo Fisher Scientific, Waltham, MA, USA) and measured by FCM flow cytometer (BD Bioscience, San Jose, CA, USA).
10
Annexin V-FITC/PI Apoptosis Assay
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To quantify the extent of apoptosis, cells were harvested with trypsin without EDTA, and visualized by Annexin V-FITC/propidium iodide staining according to the manufacturer's protocol (Invitrogen, Carlsbad, CA).
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