Annexin 5 fitc staining kit

Manufactured by Thermo Fisher Scientific

Sourced in Canada

The Annexin V/FITC staining kit is a laboratory tool used for the detection of apoptosis. It contains Annexin V, a protein that binds to phosphatidylserine, and FITC, a fluorescent dye. This kit enables the identification and quantification of cells undergoing programmed cell death through flow cytometric analysis.

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Annexin V-FITC staining Annexin V-FITC kit Annexin-V staining Annexin V-FITC Annexin V kit Annexin V

6 protocols using annexin 5 fitc staining kit

miR-29b Modulates Trophoblast Apoptosis

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The effects of miR-29b on human trophoblasts cells apoptosis were analyzed with by annexin V-FITC staining kit (Invitrogen, Carlsbad, CA USA) according to the manufacturer's instructions. Briefly, HTR8/SVneo cells were harvested after 48 h transfection of 50 nM miR-29b mimic, miRNA mimic negative control, miR-29b inhibitor or miRNA inhibitor negative control, respectively. Each sample added 5 μL Alexa Fluor® 488 annexin V and 1 μL PI. After 15 min of incubation, each sample added 400 μL 1 × annexin V-binding buffer. The samples were detected at a rate of 8,000 events per second by flow cytometry (BD Biosciences, San Jose CA, USA). The experiment was repeated for three times (n = 3).

Sun D.G., Tian S., Zhang L., Hu Y., Guan C.Y., Ma X, & Xia H.F. (2020). The miRNA-29b Is Downregulated in Placenta During Gestational Diabetes Mellitus and May Alter Placenta Development by Regulating Trophoblast Migration and Invasion Through a HIF3A-Dependent Mechanism. Frontiers in Endocrinology, 11, 169.

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Apoptosis Induction Assessment

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Cells (0.5 × 10⁶) seeded in 6-cm dishes were pre-treated (or not pre-treated) with 50 nM 8-CPT-cAMP for 30 min, then treated (or untreated) with 50 mIU/ml of l-asparaginase or 0.5 μg/ml of methadone for 12 h. After harvesting, cells were washed twice with 1× PBS by centrifugation at 1000 rpm for 5 min, then resuspended in 100 μl of staining buffer (from the Annexin V-FITC staining Kit, Invitrogen) and stained by adding 2 μl of Annexin V-FITC and 2 μl of propidium iodide (PI). Stained cells were subsequently analyzed by flow cytometry (Attune NxT flow cytometer, ThermoFisher Scientific, USA).

Lee J.K., Wang X., Wang J., Rosales J.L, & Lee K.Y. (2024). PKA inhibition kills l-asparaginase-resistant leukemic cells from relapsed acute lymphoblastic leukemia patients. Cell Death Discovery, 10, 257.

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Erlotinib and Luteolin Cytotoxicity Assay

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Erlotinib was purchased from Cayman Chemical Company (Ann Arbor, MI). Luteolin was purchased from Indofine Chemical Company (Hillsborough, NJ). Primary antibodies to Akt, ERK1/2, mTOR, p70S6K, PARP, and Bcl-xL were purchased from Cell Signaling Technology (Danvers, MA). Tubulin antibody was purchased from Sigma (St. Louis, MO). MTT assay kits were obtained from Promega (Madison, WI). DMEM and penicillin/streptomycin antibiotics were purchased from Gibco (Carlsbad, CA). Annexin V/FITC staining kit was purchased from Thermo Fisher (Waltham, MA).

Powe E., Parschauer D., Istifan J., Lin S., Duan H., Gryka R., Jean-Louis D., Tiwari A.K, & Amos S. (2022). Luteolin enhances erlotinib’s cell proliferation inhibitory and apoptotic effects in glioblastoma cell lines. Frontiers in Pharmacology, 13, 952169.

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Calcium Homeostasis and Apoptosis Signaling

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The Opti-MEM reduced serum media, fetal bovine serum, penicillin–streptomycin, Fura-2 AM, Fluo-4 AM, Mag-Fluo-4 AM, and the Annexin V-FITC staining Kit were from ThermoFisher Scientific (Burlington, ON, Canada). l-asparaginase (ASNase, ab73439) and 2,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) were from Abcam (Toronto, ON, Canada). 2,5-Di-tert-butylhydroquinone (TBHQ) and tetracaine (Tet) were from Sigma (Oakville, ON, Canada). Xestospongin-C (XeC) was from Bio-Techne (Oakville, ON, Canada). Antibodies to OPRM1 (D-12), Bid (B-3), caspase-3 (E-8), caspase-12 (1611), poly(ADP-ribose) polymerase-1 (PARP1, F-2), GAPDH (0411), calpain-1 (6C-12), cytochrome C (H-104), voltage-dependent anion-selective channel 1 (VDAC-1, B-6), tubulin (D-10) and actin (I-19) were from Santa Cruz Biotech. (Dallas, TX, USA). The antibody against cleaved caspase-3 (D1–75) was from Cell Signaling (Whitby, ON). Trypan blue was from Life Technologies (Burlington, ON, Canada). Alamar blue was from Life Technologies (ON, Canada). Calpeptin was from Calbiochem, CA, USA.

Lee J., Rosales J.L., Byun H.G, & Lee K.Y. (2021). d,l-Methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+]i. Scientific Reports, 11, 1009.

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Mitochondrial Function Assay Protocol

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RPMI 1640 media, fetal bovine serum, penicillin-streptomycin, Mag-Fluo-4 AM, Rhod-2 AM, Fluo-4 AM, Annexin V-FITC staining kit, Image-IT live mitochondria permeability transition pore assay kit, MitoSOX Red, MitoTracker green and DCFDA were from Thermo Fisher Scientific (Burlington, ON, Canada). L-asparaginase (ab73439) was from Abcam (Toronto, ON, Canada). 2,5-di-tert-butylhydroquinone (TBHQ) was from Sigma (Oakville, ON, Canada). Xestospongin-C (XeC), ruthenium red (RuR), and cyclosporine A (CsA) were from Bio-Techne (Oakville, ON, Canada). HAP1 (D-12) and actin (I19) antibodies, and Mito-Tempo were from Santa Cruz Biotech. (Dallas, TX, United States of America).

Lee J.K., Rosales J.L, & Lee K.Y. (2023). Requirement for ER-mitochondria Ca2+ transfer, ROS production and mPTP formation in L-asparaginase-induced apoptosis of acute lymphoblastic leukemia cells. Frontiers in Cell and Developmental Biology, 11, 1124164.

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Annexin V-FITC Apoptosis Assay for HCMs

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Apoptosis was analyzed through dual staining with the Annexin V-FITC/staining kit (ThermoFisher). According to the manufacturer's instructions, Annexin V-FITC and PI were added to the cellular suspension for staining, FACSCalibur flow cytometer in CellQuest 3.0.1 software (BD Biosciences, USA) was used to analyze HCMs. Percentages of apoptosis HCMs were detected by dual-color analysis.

Yang G, & Lin C. (2020). Long Noncoding RNA SOX2-OT Exacerbates Hypoxia-Induced Cardiomyocytes Injury by Regulating miR-27a-3p/TGFβR1 Axis. Cardiovascular Therapeutics, 2020, 2016259.

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