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Annexin 5 apoptosis kit

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The Annexin V apoptosis kit is a laboratory product designed to detect and quantify apoptosis, a regulated form of cell death. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a molecule that is exposed on the surface of cells undergoing apoptosis. The kit also includes a nuclear stain to identify late-stage apoptotic or necrotic cells. The core function of the Annexin V apoptosis kit is to provide a tool for researchers to study and analyze the process of programmed cell death.

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Spelling variants of this product

Annexin V (493 citations) Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (38 citations) Annexin V kit (26 citations) Annexin V Apoptosis Detection Kit eFluor 450 (10 citations) ApoTarget™Annexin-V FITC Apoptosis kit (8 citations) Dead Cell Apoptosis Kits with Annexin V AlexaFluor® 488 (4 citations) Cells Annexin V Apoptosis Detection Kits (2 citations) Dead Cell Apoptosis Kits with Annexin V (2 citations) Tali™ apoptosis assay kit/Annexin V Alexa Fluor® 488 (2 citations) Annexin V-fluorescein Isothiocyanate (FITC)/PI Cell Apoptosis Kit (2 citations)

12 protocols using annexin 5 apoptosis kit

1

Apoptosis Assay for Breast Cancer Cells

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The MCF-7 and MDA-MB-231 cells were treated with different concentrations of SB743921 for 24 h. Both nonadherent and adherent cells were collected and washed. Then, cells were stained using the Annexin V Apoptosis Kit (eBioscience, San Diego, California, USA) according to the manufacturer’s instructions. Briefly, 1×106 cells were resuspended in 100 μl of 1× binding buffer with 5 μl Annexin V-FITC. After incubation at room temperature for 20 min, samples were stained by propidium iodide and detected by flow cytometry within 1 h.
Zhu L., Xiao F., Yu Y., Wang H., Fang M., Yang Y., Sun H., Wang L, & Sheng Y. (2016). KSP inhibitor SB743921 inhibits growth and induces apoptosis of breast cancer cells by regulating p53, Bcl-2, and DTL. Anti-Cancer Drugs, 27(9), 863-872.
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2

Proliferation and Apoptosis Assays

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KPf/fC or FG cells were infected with shRNA and sorted 72 h later; 50,000 transduced cells were plated in a 24-well plate in 10% DMEM. For BrdU analysis, 24 h after plating, media was refreshed with media containing BrdU (BD Biosciences); after an 18 h pulse in BrdU-containing media, cells were trypsinized, fixed, permeabilized, and stained with anti-BrdU-APC using the BrdU flow cytometry kit (BD Biosciences). For Annexin V analysis, cells were trypsinized and analyzed with the Annexin V apoptosis kit (eBioscience) 48 h after plating.
Ferguson L.P., Gatchalian J., McDermott M.L., Nakamura M., Chambers K., Rajbhandari N., Lytle N.K., Rosenthal S.B., Hamilton M., Albini S., Wartenberg M., Zlobec I., Galván J.A., Karamitopoulou E., Vavinskaya V., Wascher A., Lowy A.M., Schürch C.M., Puri P.L., Bruneau B.G., Hargreaves D.C, & Reya T. (2023). Smarcd3 is an epigenetic modulator of the metabolic landscape in pancreatic ductal adenocarcinoma. Nature Communications, 14, 292.
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3

Characterization of PBMC and CD4+ T Cell

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PBMCs were isolated using HiSep™ LSM 1077 (Himedia Laboratories). Briefly, EDTA blood was diluted 1:1 with sterile PBS and layered on HiSep™ LSM 1077. Samples were centrifuged for 30 min at 500g without applying a brake. Comparable viability of PBMCs in control and T2DM patients was flow cytometrically determined by AnnexinV apoptosis kit (eBiosciences) (Supplementary Figure 1). The PBMC interface was carefully removed and washed twice with PBS. CD4+ T cells were purified from PBMC by standard MACS protocol (MiltenyiBiotec). Finally, PBMC and CD4+ T cells were cultured in anti-CD3 (eBioscience) coated plates treated with 2 μg/ml anti-CD28 antibodies (BD Pharmingen) in 10% FBS containing RPMI media. For DPP4 and KLK5 secretion analyses, culture supernatants were assayed for DPP4 and KLK5 levels by ELISA (R&D Systems). To identify the specific proteolytic enzyme involved in DPP4 shedding, 10 μM MMP inhibitor (GM 6001; Sigma) and 100 μg/ml KLK inhibitor (aprotinin; Sigma) were used for 24 h and 16 h respectively.
Nargis T., Kumar K., Ghosh A.R., Sharma A., Rudra D., Sen D., Chakrabarti S., Mukhopadhyay S., Ganguly D, & Chakrabarti P. (2017). KLK5 induces shedding of DPP4 from circulatory Th17 cells in type 2 diabetes. Molecular Metabolism, 6(11), 1529-1539.
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4

Quantifying Apoptosis via Annexin V Flow

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Apoptotic cells were measured using the Annexin V apoptosis kit according to the manufacturer's instructions (88-8007, eBioscience). Briefly, cells were trypsinized, washed once with PBS, and centrifugated at 1300 rpm for 5 min. After that, cells were washed once in 1×binding buffer and resuspended in 1×staining buffer to 106-107cells/ml. Then, 100μl cell (about 5×105 cells) were incubated with 10μlAnnexin V-APC at room temperature for 15 min in the dark. Finally, annexin V-stained cells were analyzed with flow cytometer (Millipore, Billerica, MA) as a measure of cell apoptosis.
Qin J., Wang W., An F., Huang W, & Ding J. (2019). PSMC2 is Up-regulated in Pancreatic Cancer and Promotes Cancer Cell Proliferation and Inhibits Apoptosis. Journal of Cancer, 10(20), 4939-4946.
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5

Cell Viability and Apoptosis Assay

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At the indicated time point after transfection, cells were collected by centrifugation after incubation with 5.0 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) Dimethyl sulfoxide (200 μl) was added into the sediments, and then spectrophotometry at 490 nm was used for the measurement of the absorbance. To identify cell apoptosis, cells were also stained using an annexin V apoptosis kit (eBiosciences, USA) and analyzed using flow cytometry.
For colony formation capacity detection, glioblastoma cells were plated in six-well plates in triplicate (400 cells/well). The cells were incubated for 2 weeks, and the culture media was refreshed per 3d. The colonies were stained with crystal violet. The colonies including 50 cells or more cells were counted.
Wang X., Cao Q., Shi Y., Wu X., Mi Y., Liu K., Kan Q., Fan R., Liu Z, & Zhang M. (2021). Identification of low-dose radiation-induced exosomal circ-METRN and miR-4709-3p/GRB14/PDGFRα pathway as a key regulatory mechanism in Glioblastoma progression and radioresistance: Functional validation and clinical theranostic significance. International Journal of Biological Sciences, 17(4), 1061-1078.
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6

Cell Cycle and Apoptosis Analysis

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The transfeted cells were stained with propidium iodide for the cycle test plus a DNA reagent kit (BD Biosciences, USA) and then measured by flow cytometry (Mindray, China). The cells ratios in the G1, S, and G2 phases were counted and compared. To identify cell apoptosis, cells were also stained using an annexin V apoptosis kit (eBiosciences, USA) and analyzed using flow cytometry.
Cao Q., Shi Y., Wang X., Yang J., Mi Y., Zhai G, & Zhang M. (2019). Circular METRN RNA hsa_circ_0037251 Promotes Glioma Progression by Sponging miR-1229-3p and Regulating mTOR Expression. Scientific Reports, 9, 19791.
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7

Quantifying Apoptosis and Caspase Activity

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Apoptotic cells were assessed using the Annexin V apoptosis kit (88–8007; eBioscience). In short, cells were trypsinized, washed and centrifuged at 283 × g for 5 min. Then, 1X binding buffer was used to wash the cells again by resuspending the cells in 200 µl. Then, the cells were incubated with 10 µl Annexin V-APC at room temperature for 15 min in the dark. Finally, the Annexin V-stained cells were analyzed with a flow cytometer (Millipore) to determine the proportion of apoptotic cells.
Caspase-3/7 are central effector caspases in apoptosis and are usually used to measure apoptotic activities. GBM cells were first transfected with the ZWINT shRNA and NC. Next, 1×104 infected cells/well were seeded in a 96-well plate. Caspase-Glo 3/7 reagent (100 µl, G8091; Promega) was added to each well, the plate was shaken for 30 min constantly and incubation was carried out at ambient temperature for 2 h. The luminescence signal was detected with an M2009PR (Tecan Infinite) plate reader.
Yang L., Han N., Zhang X., Zhou Y., Chen R, & Zhang M. (2020). ZWINT: A potential therapeutic biomarker in patients with glioblastoma correlates with cell proliferation and invasion. Oncology Reports, 43(6), 1831-1844.
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8

Quantifying Monocyte-Cancer Cell Interactions

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For cell-cell binding assays, RAW267.4 monocytes pre-labeled with Cell Tracker Red CMTPX Dye (Invitrogen, Carlsbad, CA) were co-cultured with confluent EpRAS cancer cells pre-treated with E-Cig 0.5% at a density of 2.5 × 105 cells/cm2 for 75 min to allow cell-cell binding. After the incubation, the co-culture was washed 3 times with 1X PBS to remove unbound monocytes, and fixed with 4% paraformaldehyde, followed by Hoechst 33342 staining. After three-washes, cells were visualized on fluorescent microscope. For inhibition study, RAW267.4 cells were pre-treated with 25 nM BIO 5192 (Tocris, Bristol, UK) for 4 h before co-cultured with EpRAS cells. For survival assays, the co-culture of non-labeled RAW267.4 monocytes and EpRAS cancer cells was further incubated with 100 ng/mL recombinant TRAIL (PeproTech Inc., Rocky Hill, NJ) to trigger apoptosis. After overnight treatment, the cells were washed 3 times with 1X PBS and harvested by Accutase solution. The single cell suspension was stained with anti-mouse CD45 antibody (BD Biosciences, San Jose, CA) and Annexin V apoptosis kit (Invitrogen, Carlsbad, CA) as per manufacturer’s instructions. The stained cells were analyzed with the LSR II flow cytometer (BD Biosciences, San Jose, CA). All experiments were independently repeated at least 2 times.
Pham K., Huynh D., Le L., Delitto D., Yang L., Huang J., Kang Y., Steinberg M.B., Li J., Zhang L., Liu D., Tang M.S., Liu C, & Wang H. (2020). E-cigarette promotes breast carcinoma progression and lung metastasis: Macrophage-tumor cells crosstalk and the role of CCL5 and VCAM-1. Cancer letters, 491, 132-145.
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9

Quantifying Apoptosis and Necrosis

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Cells were seeded in 100 mm culture plates and incubated overnight at 37 °C and then incubated with or without RTA 408 (1–100 nM) for 24 h followed by 200 μM H2O2 treatment for 6 h. After that, the cells were trypsinized and stained with annexin V and PI using annexin V apoptosis Kit (Invitrogen, Grand island, NY) according to the manufacturer’s protocol. The stained cells were then analyzed by flow cytometer (FC500, Beckman Colter, Indianapolis, IN) to differentiate among viable (annexin V−/PI−), early apoptotic (annexin V+/PI−), late apoptotic (annexin V+/PI+) cells, and necrotic cells (annexin V−/PI+).
Liu X., Ward K., Xavier C., Jann J., Clark A.F., Pang I.H, & Wu H. (2015). The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation. Redox Biology, 8, 98-109.
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10

Flow Cytometry Analysis of Apoptosis

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Flow cytometry was performed as previously described18 (link). Cells were harvested at various time points after they were harvested with trypsin-EDTA, washed with PBS, and fixed in 70% ethanol at 4 °C for 1 h. Ethanol was removed, and the cell pellet was washed with PBS twice before staining. An Annexin-V apoptosis kit (Invitrogen) was used for the detection of cell apoptosis using a BD FACS Celesta flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, propidium iodide (PI) fluorescence was analyzed by excitation with a 488 nm argon ion laser and detection using a 620 nm band-pass filter. Doublets and higher aggregates were excluded by gating, and 10,000 events (excluding doublets) were recorded per sample.
He J.G., Li H.R., Han J.X., Li B.B., Yan D., Li H.Y., Wang P, & Luo Y. (2018). GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes. Scientific Reports, 8, 9047.
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