Annexin 5 and 7 aad

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Annexin V is a protein that binds to phosphatidylserine, a phospholipid that is exposed on the surface of apoptotic cells. 7-AAD (7-aminoactinomycin D) is a fluorescent dye that binds to DNA and is often used to detect cell death or membrane permeability.

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Lab products found in correlation

Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions) Cyan adp, by Beckman Coulter (2 mentions) Moflo xdp, by Beckman Coulter (2 mentions) Facscanto 2, by BD (1 mentions) Countbright absolute cell counting beads, by Thermo Fisher Scientific (1 mentions) Trypan blue, by Thermo Fisher Scientific (1 mentions) Facsverse cytometer, by BD (1 mentions) Tcrβ h57 597, by BioLegend (1 mentions) Adam10, by R&D Systems (1 mentions) White 96 well plate, by Corning (1 mentions) Caspase glo 3 7 assay, by Promega (1 mentions) Kaluza analysis 1, by Beckman Coulter (1 mentions) Cell lab quanta sc flow cytometer, by Beckman Coulter (1 mentions) Guavasoft 2, by Merck Group (1 mentions) Guava easycyte 8ht, by Merck Group (1 mentions) Cell staining buffer, by BioLegend (1 mentions) Lsrii fortessa, by BD (1 mentions) Facsymphony, by BD (1 mentions) Anti cd45, by BioLegend (1 mentions) Anti granzyme b, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

7-AAD (474 citations) Annexin V (328 citations) Annexin V-APC and 7-AAD (8 citations) Annexin V-FITC and 7-AAD (5 citations) FITC-conjugated Annexin V and 7-AAD (3 citations) APC Annexin V Apoptosis Detection Kit and 7-AAD (2 citations) Annexin V and 7-aminoactinomycin D (7-AAD) (2 citations) FITC annexin V and 7-AAD apoptosis detection kit (2 citations)

14 protocols using annexin 5 and 7 aad

Quantifying Neutrophil Apoptosis in Lamina Propria

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The absolute number of neutrophils in the lamina propria (LP) was calculated using CountBright Absolute Cell Counting Beads (Thermo Scientific) according to the manufacturer’s instructions.
To evaluate apoptosis, staining with annexin V and 7-AAD (BioLegend) was performed on purified neutrophils pre-treated or not with 100 U/ml of IFN-λ or IFN-β and stimulated with 10 μg/ml LPS for 3 h.
To evaluate apoptosis on LP neutrophils, LP cells were stained for CD45, Ly6G and CD11b (antibodies identified above (‘Reagents and antibodies’); 1:200 dilution) and were subsequently stained with annexin V and 7-AAD (Cat. No. 640930, BioLegend) according to the manufacturer’s instruction.
All samples were analyzed with a BD FACSCanto II.

Broggi A., Tan Y., Granucci F, & Zanoni I. (2017). IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function. Nature immunology, 18(10), 1084-1093.

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Sorting and Characterizing Hematopoietic Stem Cells

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Cells were suspended for cell sorting in Hanks’ balanced salt solution (HBSS) containing 5%(vol/vol) fetal bovine serum (FBS) and 2 mM EDTA as previously described²⁷. The following antibodies were used to define lineage positive cells: 145-2C11 (CD3ε), GK1.5 (CD4), 53–6.7 (CD8), RB6-8C5 (Ly-6G/Gr1), M1/70 (CD11b/Mac-1), TER119 (Ly-76/TER119), 6B2 (CD45R/B220), and eBio1D3 (CD19). Red blood cells were lysed with RBC Lysis Buffer (eBioscience) before staining for lineage markers. For the Lin⁻ Sca-1⁺ cKit⁺ (LSK) bone marrow cell sorting, the antibodies 2B8 (cKit/CD117) and D7 (Sca-1/Ly-6A/E) antibodies were also used. To determine donor-derived chimerism in the transplantation-based assays, peripheral blood from the recipients was obtained by the submandibular bleeding method and prepared for analysis as previously described^{20 (link)}. All antibodies were purchased from eBioscience. Apoptosis assays were performed by staining cells with Annexin V and 7-AAD (BioLegend). Cell cycle status was analyzed by staining cells with 2.5 μg/ml PI containing 0.1% BSA and 2 μg/ml RNase after fixation with 70% ethanol. Flow cytometric analysis and cell sorting were carried out on the Moflo XDP, Cyan ADP (Beckman Coulter) or S3 (Bio-Rad), and the data were analyzed with FlowJo software (Tree Star Inc.).

Hattori A., Tsunoda M., Konuma T., Kobayashi M., Nagy T., Glushka J., Tayyari F., McSkimming D., Kannan N., Tojo A., Edison A.S, & Ito T. (2017). Cancer progression by reprogrammed BCAA metabolism in myeloid leukemia. Nature, 545(7655), 500-504.

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Immature DCs Modulation by Cannabinoids

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For titration experiments, immature hmoDCs from healthy donors (10⁶ cells/mL) were treated with LPS (0.1 μg/mL) plus different doses of WIN55212-2, HU210 or HU308 (1, 5, 10 and 20 μM) or with TLR2L (25 ng/mL) or MFs (25 ng/mL IL-1β and 50 ng/mL TNFα) plus different doses of WIN55212-2 (5, 10 and 20 μM).
Immature hmoDCs or human total blood DCs from healthy donors (10⁶ cells/mL) were stimulated with medium (unstimulated), WIN55212-2 (10 µM), LPS (0.1 μg/mL) or LPS plus WIN55212-2 for 18 h. Cells were used to analyse their phenotype by flow cytometry and cell‐free supernatants to quantify IL-8, IL‐1β, IL‐6, TNFα and IL‐10 by sandwich enzyme-linked immunosorbent assay (ELISA). For inhibition experiments, hmoDCs were preincubated for 1 h with Rimonabant (20 µM), AM630 (20 µM), GW6471 (25 µM), GW9662 (10 µM) or 3-MA (25 µM) or corresponding vehicle controls prior to activation. Then, cells were stimulated with LPS plus WIN55212-2 for 18 h in the presence of the corresponding inhibitors. Cell viability was analysed in all the cases by trypan blue (Gibco) exclusion and/or Annexin V and 7-AAD (Biolegend) by flow cytometry analysis.

Angelina A., Pérez-Diego M., López-Abente J., Rückert B., Nombela I., Akdis M., Martín-Fontecha M., Akdis C, & Palomares O. (2021). Cannabinoids induce functional Tregs by promoting tolerogenic DCs via autophagy and metabolic reprograming. Mucosal Immunology, 15(1), 96-108.

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Annexin V and 7-AAD Apoptosis Assay

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Cells treated with DMSO or OSSL_325096 for 48‐72 hours were washed with annexin V buffer, stained with annexin‐V and 7‐AAD (BioLegend, San Diego, CA, USA), and analyzed using a FACSVerse cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Nishimura N., Radwan M.O., Amano M., Endo S., Fujii E., Hayashi H., Ueno S., Ueno N., Tatetsu H., Hata H., Okamoto Y., Otsuka M., Mitsuya H., Matsuoka M, & Okuno Y. (2019). Novel p97/VCP inhibitor induces endoplasmic reticulum stress and apoptosis in both bortezomib‐sensitive and ‐resistant multiple myeloma cells. Cancer Science, 110(10), 3275-3287.

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Multiparametric Immune Cell Profiling

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Anti-mouse CD16/CD32 (Clone 2.4G2), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), MHC II (M5/114.15.2), F4/80 (BM8), Clec4F (3E3F9), Tim4 (RMT4-54), CD11b (M1/70), Ly6C (HK1.4), TCR-β (H57-597), NK1.1 (PK136), CD4 (GK1.5), CD8 (53–6.7), CXCR6 (SA051D1), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), IFN-γ (XMG1.2), as well as Annexin V and 7-AAD were purchased from Biolegend. Anti-mouse CRIg (NLA14), Nr4a1 (12.14) were purchased from ThermoFisher Scientific. CXCL16 (12–81) was purchased from BD Biosciences. ADAM10 (139712) was purchased from R&D Systems.

Liu G., Abas O., Strickland A.B., Chen Y, & Shi M. (2021). CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection. PLoS Pathogens, 17(10), e1009968.

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Caspase-3/7 and Apoptosis Assays

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Cells were treated with a 1:4 dilution series ranging from 20 µM to 0.078 µM LMK-235 in 96-well cell polystyrene cell culture plates (Greiner Bio-One). Caspase-Glo 3/7 assay (Promega, Mannheim, Germany) was performed after 0, 8, 24, and 32 h according to the manufacturer’s protocol. Supernatants including the reaction mixture were transferred for luminescence measurements into white 96-well plates (Corning, Kaiserslautern, Germany). Luminescence measurements were performed on the Infinity M200 reader with an integration time of 5 s.
After 24 h incubation with a LMK-235 1:4 dilution series (0.078–20 µM) in 6-well polystyrene cell culture plates (TPP), cells were harvested and stained according to the manufacturer’s instructions with Annexin-V and 7-AAD (both BioLegend, Koblenz, Germany) for FACS analysis of apoptosis on a Cell Lab Quanta SC flow cytometer (Beckman-Coulter, Vienna, Austria) and the Kaluza Analysis 1.3 software (Beckman-Coulter).

Wanek J., Gaisberger M., Beyreis M., Mayr C., Helm K., Primavesi F., Jäger T., Di Fazio P., Jakab M., Wagner A., Neureiter D, & Kiesslich T. (2018). Pharmacological Inhibition of Class IIA HDACs by LMK-235 in Pancreatic Neuroendocrine Tumor Cells. International Journal of Molecular Sciences, 19(10), 3128.

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Apoptosis Quantification in Chlamydia-Infected DCs

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The level of apoptosis in chlamydia pulsed and nonpulsed WT and ENO1 knockdown DCs was determined using Annexin V and 7-AAD flow cytometry (Biolegend, San Diego, CA). The cells were then collected at time 0 and 2 h after infection and washed with cold BioLegend Cell Staining Buffer twice and then resuspended in Annexin V binding buffer. Five microliters of fluorescein isothiocyanate (FITC)-Annexin V and 5 μl of 7-amino-actinomycin D (7-AAD), viability staining solution were added to 100 μl cell suspensions and incubated in the dark at room temperature for 15 min. 400 μl of binding buffer was added to the sample and the mixture was vortexed and then analyzed by flow cytometry using a guava easyCyte 8HT (EMD Millipore, Billerica, MA). For each sample, at least 100,000 events were collected. Data was analyzed with guavaSoft 2.7 (EMD Millipore, Billerica, MA).

Ryans K., Omosun Y., McKeithen D.N., Simoneaux T., Mills C.C., Bowen N., Eko F.O., Black C.M., Igietseme J.U, & He Q. (2017). The immunoregulatory role of alpha enolase in dendritic cell function during Chlamydia infection. BMC Immunology, 18, 27.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry was carried out on a LSR II Fortessa or a FACSymphony (BD Bioscience) with anti‐phospho‐Histone H2A.X Monoclonal (MA537049, Thermo Fisher), anti‐CD45 (1033132, BioLegend), anti‐CD3 (100222, BioLegend), anti‐NK1.1 (108748, Biolegend), anti‐NKp46 (137606, BioLegend), anti‐CD107a (560648, BD Biosciences), anti‐Granzyme B (812‐8898, Invitrogen), anti‐Interferon‐gamma (554413, BD), and Annexin V and 7AAD (BioLegend, 640934).

Pelletier A., Nelius E., Fan Z., Khatchatourova E., Alvarado‐Diaz A., He J., Krzywinska E., Sobecki M., Nagarajan S., Kerdiles Y., Fandrey J., Gotthardt D., Sexl V., de Bock K, & Stockmann C. (2023). Resting natural killer cell homeostasis relies on tryptophan/NAD + metabolism and HIF‐1α. EMBO Reports, 24(6), e56156.

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Modulation of NK Cell Cytotoxicity

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PBMCs were cultured in RPMI 1640 (Lonza) supplemented with 2 mM l-glutamine, 2% heat-inactivated fetal calf serum (Gibco), penicillin (100 IU/ml), streptomycin (100 µg/ml), and low-dose IL-2 (2 U/ml; Sigma) in the presence of vehicle (<0.1% ethanol), dexamethasone (1 µM; Sigma), LXA₄ (50 nM; Cayman Chemical), or both dexamethasone and LXA₄ for 48 hours. dexamethasone and LXA₄ were redosed after 24 hours. After 48 hours, PBMCs were washed and placed into coculture with K562 myeloid tumor cells that were labeled with the DNA dye eFluor 670 (eBioscience) at an effector/target ratio of 20:1 (PBMC/K562) for 4 hours at 37°C in 5% CO₂. NK cell–mediated lysis of target cells was then assessed by gating on eFluor 670⁺ K562 cells and using annexin V and 7-AAD (BioLegend) staining to identify necrotic/ late apoptotic cells. To determine the cellular source of cytotoxic mediators and cytokines in coculture experiments, we exposed PBMCs to K562 cells at an effector/target ratio of 20:1 for 2 hours followed by 4 hours in the presence of GolgiStop (BD). Cells were then stained as described above, and intracellular perforin and IL-17 were measured in NK cells, CD8⁺ T cells, or CD4⁺ T cells by flow cytometry.

Duvall M.G., Barnig C., Cernadas M., Ricklefs I., Krishnamoorthy N., Grossman N.L., Bhakta N.R., Fahy J.V., Bleecker E.R., Castro M., Erzurum S.C., Gaston B.M., Jarjour N.N., Mauger D.T., Wenzel S.E., Comhair S.A., Coverstone A.M., Fajt M.L., Hastie A.T., Johansson M.W., Peters M.C., Phillips B.R., Israel E, & Levy B.D. (2017). Natural killer cell–mediated inflammation resolution is disabled in severe asthma. Science immunology, 2(9), eaam5446.

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Sorting and Characterizing Hematopoietic Stem Cells

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