Annexin 5 buffer

Manufactured by BD

Sourced in United States, United Kingdom

Annexin V buffer is a solution used in flow cytometry and other cell-based assays to detect and quantify apoptotic cells. It provides a stable environment for the binding of Annexin V, a calcium-dependent phospholipid-binding protein, to phosphatidylserine residues exposed on the surface of cells undergoing programmed cell death.

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Lab products found in correlation

7 aad, by BD (3 mentions) Cytoflex, by Beckman Coulter (2 mentions) Guava easycyte flow cytometer, by Merck Group (2 mentions) Incyte software, by Merck Group (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions) Mgcl2, by Merck Group (2 mentions) Nhp01srm, by BioIVT (2 mentions) Phycoerythrin conjugated anti c3b antibodies, by BioLegend (2 mentions) Facsymphony a3 cytometer, by BD (2 mentions) Ghost dye red 780 viability dye, by Cytek Biosciences (2 mentions) Aqua live dead amine dye amcyan, by Thermo Fisher Scientific (2 mentions) Annexin 5 fitc, by BioLegend (2 mentions) Anti cd8 apc, by BD (1 mentions) Anti cd45 apc cy7, by BD (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions) Annexin 5 pe 7 aad, by BD (1 mentions) Canto 2 flow cytometer, by BD (1 mentions) Tryple express, by BD (1 mentions) Cell processing tubes, by BD (1 mentions)

Close spelling variants of this product

Annexin V (870 citations) Annexin V binding buffer (445 citations) Annexin V staining buffer (10 citations) Annexin buffer (9 citations) Annexin V binding buffer 1X (4 citations) Annexin V-FITC binding buffer (4 citations) Annexin V binding buffer 1×, (3 citations) FITC-conjugated Annexin V and Annexin V binding buffer (2 citations) Annexin V binding buffer (pH 7.4) (2 citations) 1 × Annexin V-binding buffer solution (1 citations)

26 protocols using annexin 5 buffer

Organoid Killing Assay for Immunogenic Peptide-Specific T Cells

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PBMCs were incubated with immunogenic peptide pools for nine days with three cycles of stimulations. After stimulation, PBMCs were washed and stained with anti CD3 AF700, anti CD8 APC, and Propidium iodide (PI, BD). Peptide specific live T cells (CD3⁺CD8⁺PI⁻) were FACS isolated for organoid killing assay. Organoids were dissociated to single cells with Tryple Express, labeled with CFSE (BD), and cocultured in at least triplicate with sorted T cells at a 10:1 effector:target ratio in T cell culture medium. To analyze the activity of ICIs, 5 µg mL⁻¹ PD‐1 inhibitor and/or 10 µg mL⁻¹ CTLA‐4 inhibitor was added during the progress of stimulation and killing assay. Each organoid killing assay was repeated twice. After 3 days of coculture, organoids and T cells were collected. Cells were washed in FACS buffer and stained with anti CD45 APC‐Cy7, Annexin V PE, 7‐AAD (BD) in Annexin V buffer (BD) for 15 min at RT. The apoptosis of organoids labeled with CFSE were analyzed with flow cytometer Canto II (BD) or CytoFLEX (Beckman). FITC⁺CD45 APC‐Cy7⁻ was used to detect organoids and Annexin V⁻7‐AAD⁻ was used to define live cells.

Wang W., Yuan T., Ma L., Zhu Y., Bao J., Zhao X., Zhao Y., Zong Y., Zhang Y., Yang S., Qiu X., Shen S., Wu R., Wu T., Wang H., Gao D., Wang P, & Chen L. (2022). Hepatobiliary Tumor Organoids Reveal HLA Class I Neoantigen Landscape and Antitumoral Activity of Neoantigen Peptide Enhanced with Immune Checkpoint Inhibitors. Advanced Science, 9(22), 2105810.

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Isolation and Quantification of Extracellular Vesicles

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Blood samples were collected in lavender EDTA vacutainer tubes (BD, Oxford, UK). Platelet poor plasma (PPP) was obtained by double centrifugation at 5000g for 5 min as previously described and stored in 100 μl aliquots at −80°C until required.²⁴ MVs were then isolated from PPP after centrifugation at 17,000g for 1 h at 4°C. Annexin‐V conjugated to fluorescein isothiocyanate (FITC) that was diluted in annexin‐V buffer (BD Pharmingen) was used to identify total MVs using flow cytometry.²⁴ 1.1 μm latex beads were used to set the upper threshold on forward scatter to distinguish maximum MVs size. MVs captured in this way were defined as annexin‐V+ co‐expressing‐specific cell surface markers, determined by using appropriate isotype control antibodies for each marker. MVs were enumerated in a standardised fashion by using the proportion of a fixed number of 3 μm latex beads counted and the volume of sample from which the MVs were analysed (Figure S3).

Eddama M.M., Gurung R., Fragkos K., Lorgelly P., Cohen R., Loizidou M, & Clapp L. (2022). The role of microvesicles as biomarkers in the screening of colorectal neoplasm. Cancer Medicine, 11(15), 2957-2968.

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Oxidative Stress-Induced Cell Death in ARPE-19 Cells

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ARPE-19 cells were treated with the indicated concentrations of H₂O₂ for 12 h or 0.5 mM H₂O₂ for the indicated times. The cells were harvested and stained with PI at a final concentration of 5 μg/ml. Cell death was analyzed using a Guava easyCyte flow cytometer (Millipore). In another set of experiments, H₂O₂-treated ARPE-19 cells were harvested and washed using Annexin V buffer provided by the supplier (BD Biosciences) and then stained with Annexin V. Next, PI was added at a final concentration of 5 μg/ml. The cells were then evaluated using a Guava easyCyte flow cytometer and quantified using InCyte software (Millipore).

Jang K.H., Do Y.J., Son D., Son E., Choi J.S, & Kim E. (2017). AIF-independent parthanatos in the pathogenesis of dry age-related macular degeneration. Cell Death & Disease, 8(1), e2526-.

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Rhesus Macaque PBMC Cytotoxicity Assay

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Peripheral blood mononuclear cells (PBMCs) were isolated from cell processing tubes (BD Biosciences, San Jose, CA) from 3 rhesus macaques and suspended in RPMI supplemented with 10% FBS, 1% Hepes Buffer, glutamine and penicillin/streptomycin at a concentration of 10⁶ cells/mL. LtxA was added at concentrations of 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, or 10 μg/mL, and cells were incubated for 1.5 hours at 37°C. Cells were then washed and plated in a 96-well round-bottom plate with 10⁶ cells/well. Cells were resuspended in 100 μL of Annexin V buffer (BD Biosciences) per the manufacturer’s instructions and stained with surface antibodies for 15 minutes at room temperature. The surface staining consisted of antibodies against CD3, CD4, CD8, CD56, CD11a, CD16, CD95, CD28, and CD20. The cells were then washed twice with Annexin V buffer with addition of 7AAD after the final wash and immediately acquired on the flow cytometer. Heat killed cells were used as a compensation control for 7AAD staining. For experiments using activated cells, surface staining was done prior to incubation with the same varying concentrations of LtxA, with or without addition of phorbol 12-myristate 13-acetate and ionomycin.

Samy K., Anderson D., Lo D., Mulvihill M., Song M., Farris A., Parker B., MacDonald A., Lu C., Springer T., Kachlany S., Reimann K., How T., Leopardi F., Franke K., Williams K., Collins B, & Kirk A. (2017). Selective Targeting of High-Affinity LFA-1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 17(5), 1193-1203.

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Annexin V-based Apoptosis Assay

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For analysis of Annexin V positive cells, RDES and TC-32 cells harboring a Dox-inducible shRNA against SOX6 were seeded at 3 × 10⁵ cells per 10 cm dish and treated with 0.1 µg/ml Dox every 48 h for knockdown. After 96 h, cells were washed with PBS and cells were resuspended in 1 × Annexin V buffer (BD Biosciences) with 5 µl of Annexin V and 5 µl PI solution for further 15 minutes. Analysis of Annexin V positivity was performed with BD Accuri C6 Cytometer (BD Biosciences) by counting at least 1 × 10⁵ events. An example for the gating strategy is provided in Supplementary Fig. 7b.

Marchetto A., Ohmura S., Orth M.F., Knott M.M., Colombo M.V., Arrigoni C., Bardinet V., Saucier D., Wehweck F.S., Li J., Stein S., Gerke J.S., Baldauf M.C., Musa J., Dallmayer M., Romero-Pérez L., Hölting T.L., Amatruda J.F., Cossarizza A., Henssen A.G., Kirchner T., Moretti M., Cidre-Aranaz F., Sannino G, & Grünewald T.G. (2020). Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma. Nature Communications, 11, 2423.

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Evaluating NK Cell Apoptosis with MTA

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NK cells rested overnight with 100 U/ml IL-2 were incubated with increasing concentrations of MTA in complete medium without additional IL-2 for 4 h at 37°C. Cells were harvested, washed, and labeled in 100 μl of 1 × Annexin V Binding Buffer (BD Biosciences) with 5 μl of Annexin V-FITC and 7AAD at room temperature for 15′. Afterwards, 400 μl of 1 × Annexin V buffer was added and cells were analyzed at a FACSCanto II machine (BD Biosciences). FlowJo software was used for analyzing FACS data (FlowJo LLC).

Jacobs B., Schlögl S., Strobl C.D., Völkl S., Stoll A., Mougiakakos D., Malmberg K.J., Mackensen A, & Aigner M. (2020). The Oncometabolite 5′-Deoxy-5′-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation. Frontiers in Immunology, 11, 2128.

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LCMV-activated NK cell cytotoxicity

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Activated NK cells were harvested from donor mice 2 days after LCMV WE infection and isolated using DX5⁺ magnetic beads (Miltenyi Biotech,Bergisch Gladbach, Germany). 20’000 YAC-1 cells were co-cultured with DX5⁺ NK cells in triplicates in a ratio of 1:1, 1:10 and 1:25. After 4 h incubation at 37°C, surface stainings were performed in PBS and the samples were washed. The apoptotic markers, Annexin V-PB (BioLegend UK Ltd) and 7AAD (BD Biosciences) were diluted in Annexin V Buffer (BD Biosciences) and incubated for 30 min at 4°C followed by transfer on ice and direct acquisition of the samples.

Pallmer K., Barnstorf I., Baumann N.S., Borsa M., Jonjic S, & Oxenius A. (2019). NK cells negatively regulate CD8 T cells via natural cytotoxicity receptor (NCR) 1 during LCMV infection. PLoS Pathogens, 15(4), e1007725.

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Quantifying Glioma Cell Apoptosis

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To measure apoptosis resulting from Tim-3 or Gal-9 knockdown, glioma cells were placed in 0.1% (v/v) bovine serum albumin (BSA) and stained with Annexin V (BD Biosciences). The cells were then washed with Annexin V buffer (BD Biosciences) and stained with 7-AAD (BD Pharmingen). To exclude spontaneous cell death, the % apoptosis was calculated based on the Annexin V and 7-AAD signal generated by control living glioma cells that were not treated with siRNA. Apoptosis was quantitated by flow cytometry (CytoFLEX; Beckman Coulter).

Sim J., Park J., Kim S., Hwang S., Sung K., Lee J.E., Yang S., Cho K., Lee S., Moon J.S., Ahn J, & Lim J. (2022). Association of Tim-3/Gal-9 Axis with NLRC4 Inflammasome in Glioma Malignancy: Tim-3/Gal-9 Induce the NLRC4 Inflammasome. International Journal of Molecular Sciences, 23(4), 2028.

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Evaluating TWE's Effect on RAW264.7 Viability

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To examine the effect of TWE on cell viability of RAW264.7 cells, RAW264.7 cells were seeded in 24-well plates at a cell density of 1 × 10⁶ cells/ml (2 × 10⁵ cells/well) and stimulated with either TWE (100, 300, 900, and 2700 μg/ml) or LPS (1 μg/ml) for 12 h at 37 °C in a CO₂ incubator. Total cells were washed in PBS, and 100 μl annexin V buffer (BD Pharmingen, San Diego, CA) containing 10 μl 7-amino actinomycin (7-AAD) was added for 15 min at room temperature in the dark. Cells were resuspended in 300 μl annexin V buffer and were analyzed within 1 h by flow cytometry. The relative cell viability (%) was expressed as a percentage (7-AAD negative) relative to the control cells.

Park H.J., Lee S.W., Kwon D.J., Heo S.I., Park S.H., Kim S.Y, & Hong S. (2017). Oral administration of taheebo (Tabebuia avellanedae Lorentz ex Griseb.) water extract prevents DSS-induced colitis in mice by up-regulating type II T helper immune responses. BMC Complementary and Alternative Medicine, 17, 448.

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Quantifying Complement-Mediated C3b Deposition

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Endothelial cells (50,000 cells per well) were seeded in a 96-well plate in serum dilution buffer (SDB) (1× annexin V buffer (51-66121E, BD Pharmingen), 1 mM MgCl₂ (68475, Sigma) and 1% BSA (A9576, Sigma)). Pooled normal human serum (NHS, Complement Technology) or cynomolgus monkey serum (NHP01SRM, BioIVT) diluted in SDB were added to appropriate wells at a final concentration of 25% and incubated for 30 min at 37 °C. For negative controls, cells were treated with 25% serum containing 10 mM EDTA (15575038, Thermo Fisher) to inactivate complement. After incubation, cells were washed and stained with phycoerythrin-conjugated anti-C3b antibodies at a 1:100 dilution (846104, BioLegend) and Ghost Dye Red 780 viability dye at a 1:500 dilution (13-0865, Tonbo Biosciences) for 30 min at 4 °C in the dark. Cells were washed twice, immediately acquired on a BD FACSymphony A3 cytometer and analysed in Flow Jo. C3b deposition was plotted as MFI and statistics were calculated using GraphPad Prism v8.2.0.

Anand R.P., Layer J.V., Heja D., Hirose T., Lassiter G., Firl D.J., Paragas V.B., Akkad A., Chhangawala S., Colvin R.B., Ernst R.J., Esch N., Getchell K., Griffin A.K., Guo X., Hall K.C., Hamilton P., Kalekar L.A., Kan Y., Karadagi A., Li F., Low S.C., Matheson R., Nehring C., Otsuka R., Pandelakis M., Policastro R.A., Pols R., Queiroz L., Rosales I.A., Serkin W.T., Stiede K., Tomosugi T., Xue Y., Zentner G.E., Angeles-Albores D., Chris Chao J., Crabtree J.N., Harken S., Hinkle N., Lemos T., Li M., Pantano L., Stevens D., Subedar O.D., Tan X., Yin S., Anwar I.J., Aufhauser D., Capuano S., Kaufman D.B., Knechtle S.J., Kwun J., Shanmuganayagam D., Markmann J.F., Church G.M., Curtis M., Kawai T., Youd M.E, & Qin W. (2023). Design and testing of a humanized porcine donor for xenotransplantation. Nature, 622(7982), 393-401.

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