Annexin 5 fluos staining kit

Manufactured by Roche

Sourced in Germany, Switzerland, United States, France, Japan, China, Spain, United Kingdom, Italy

The Annexin-V-FLUOS Staining Kit is a laboratory product designed for the detection and quantification of apoptosis. It provides a sensitive and reliable method for the assessment of apoptosis in cell cultures and samples. The kit contains Annexin-V-FLUOS, a fluorescent conjugate that binds to phosphatidylserine, a marker of apoptotic cells. The kit also includes propidium iodide, a dye that stains necrotic cells. This combination of reagents allows for the identification and differentiation of early apoptotic, late apoptotic, and necrotic cells.

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Spelling variants of this product

Annexin-V-FLUOS (70 citations) Annexin V (39 citations) Annexin-V-FLUOS Kit (27 citations) Annexin V kit (4 citations) FITC Annexin-V-FLUOS staining Kit (3 citations) Annexin-V staining (2 citations)

439 protocols using annexin 5 fluos staining kit

Apoptosis and γδ T cell analysis by flow cytometry

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Apoptotic lymphocytes were analyzed by flow cytometry (BriCyte E6, Mindray, Shenzhen, China) following staining with Annexin-V (FITC) and propidium iodide (PI) [21 (link)] using the Annexin-V-FLUOS staining kit (Boehringer Mannheim, GmbH, Mannheim, Germany). The cell suspensions were analyzed by flow cytometry with differentiation of 20,000 cells. The lymphocytes were distributed over three different quadrants of the dot plots, representing viable (Annexin-V−/PI−), apoptotic (Annexin-V+/PI−), and necrotic lymphocytes (Annexin-V+/PI+). The dot plots were assessed using MR Flow software (Mindray, China).
For the analysis of the γδ T cells, mouse monoclonal anti-bovine γδ TCR (T cell receptor) (GB21A, IgG2b; Serotec Ltd., Oxford, UK) antibody was used [19 (link),22 (link)]. All of the mAbs (monoclonal antibodies) were from Serotec Ltd., Oxford, UK. FITC-conjugated goat anti-mouse IgG2b (Southern Biotechnology Associated, Inc., Birmingham, AL, USA) was used as the secondary antibody [19 (link)].

Slama P., Zavadilova T., Pavlik A., Horky P., Skalickova S., Skladanka J., Roychoudhury S., Baldovska S., Kolesarova A., Konecny R., Tancin V, & Zouharova M. (2021). Effect of Streptococcus uberis on Gamma Delta T Cell Phenotype in Bovine Mammary Gland. Animals : an Open Access Journal from MDPI, 11(12), 3594.

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Flow Cytometric Analysis of Apoptotic Lymphocytes

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Apoptotic lymphocytes were analyzed by flow cytometry (FACSCalibur apparatus, Becton Dickinson, San Jose, CA, USA) following staining with Annexin-V (FITC) and propidium iodide (PI) [33 (link)]. For that purpose, we used the Annexin-V-FLUOS staining kit (Boehringer Mannheim, GmbH, Mannheim, Germany). Five hundred µL of the incubation buffer was mixed with 10 µL of FITC-Annexin-V and 10 µL of PI solution. After 15 min of incubation at room temperature, the cell suspension was analyzed by flow cytometry with differentiation of 20,000 cells. Lymphocytes were distributed over three different quadrants of dot plots representing viable (Annexin-V-/PI-), apoptotic (Annexin-V+/PI-), and necrotic lymphocytes (Annexin-V+/PI+). The percentages of apoptotic lymphocytes were calculated from the total number of lymphocytes. The dot plots were assessed using the WinMDI software (Windows Multiple Document Interface for Flow Cytometry; Purdue University, West Lafayette, IN, USA).
For analysis of CD44, mouse anti-ovine antibody CD44 BAG40A (VMRD Inc. Pullman, Washington, USA) diluted 1:50 and FITC labelled IgG3 (SouthernBiotech, Birmingham, Alabama, USA) diluted 1:100 as the primary and the secondary antibodies were used, respectively.

Slama P., Kabourkova E., Sladek Z., Zavadilova T., Kratochvilova L., Kharkevich K., Roychoudhury S., Pavlik A., Roztocilova A., Uhrincat M., Tancin V., Kimura K., Konecny R., Kiku Y., Watanabe A., Kwak J.Y, & Zouharova M. (2020). Effect of Lipopolysaccharide and Muramyl Dipeptide on Apoptosis of Bovine Mammary Gland Lymphocytes. Animals : an Open Access Journal from MDPI, 10(6), 990.

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Cell Viability and Apoptosis Assays

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The trypan blue exclusion assay has been previously described^{22 (link)} and was used for quantification of cells prior to seeding for Cell Titer Glo assays. The Cell Titer Glo assay (Promega, Madison, WI) was used for proliferation studies and carried out according to manufacturer instructions. Cell viability is reported as percentage of control (untreated) cells, and error bars represent the standard error of the mean for each data point. Programmed cell death of inhibitor-treated cells was determined using the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis, IN), as previously described^{22 (link)}. Cell cycle analysis was carried out via propidium iodide staining and FACS analysis.

Weisberg E., Nonami A., Chen Z., Nelson E., Chen Y., Liu F., Cho H., Zhang J., Sattler M., Mitsiades C., Wong K.K., Liu Q., Gray N, & Griffin J.D. (2014). Up-regulation of IGF-1R by mutant RAS in leukemia and potentiation of RAS signaling inhibitors by small molecule inhibition of IGF-1R. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(21), 5483-5495.

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Annexin-V and PI Assay for Apoptosis

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Programmed cell death of inhibitor-treated cells was determined using the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis, IN), as previously described [6 (link)]. Briefly, cells were washed once with 1X PBS and pelleted by centrifugation for 5 minutes at 1500 rpm. Cells were resuspended in 100μl of 20% propidium iodide (PI) and 20% Annexin-V-fluorescein labeling reagent, either agent alone (as controls), or were left unstained by diluting only in 1X binding buffer (as a control). All samples were incubated for 10-15 minutes at room temperature, and then stained cells were diluted in 0.8 mL of 1X binding buffer. Cells were then analyzed by flow cytometry.
Cell cycle analysis was performed as previously described [6 (link)]. Briefly, around 500,000 cells were centrifuged at 1500 rpm for 5 min and washed in 1X PBS, and the pellet was resuspended in 500 μl of propidium iodide solution (50 μg/ml propidium iodide, 0.1% NP-40, 0.1% sodium citrate). The mixture was stored in the dark at 4°C for a minimum of 15 min, and then analyzed by flow cytometry.

Weisberg E.L., Puissant A., Stone R., Sattler M., Buhrlage S.J., Yang J., Manley P.W., Meng C., Buonopane M., Daley J.F., Lazo S., Wright R., Weinstock D.M., Christie A.L., Stegmaier K, & Griffin J.D. (2017). Characterization of midostaurin as a dual inhibitor of FLT3 and SYK and potentiation of FLT3 inhibition against FLT3-ITD-driven leukemia harboring activated SYK kinase. Oncotarget, 8(32), 52026-52044.

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Cell Proliferation and Death Assays

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Cell counts for proliferation studies were obtained using the trypan blue exclusion assay, as previously described (24 (link)). Error bars represent the standard error of the mean for each data point. Programmed cell death of inhibitor-treated cells was determined using the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis, IN), as previously described (24 (link)). Cell cycle analysis was performed as previously described (24 (link)).

Weisberg E., Halilovic E., Cooke V.G., Nonami A., Ren T., Sanda T., Simkin I., Yuan J., Antonakos B., Barys L., Ito M., Stone R., Galinsky I., Cowens K., Nelson E., Sattler M., Jeay S., Wuerthner J.U., McDonough S.M., Wiesmann M, & Griffin J.D. (2015). Inhibition of wild-type p53-expressing AML by novel small molecule HDM2 inhibitor, CGM097. Molecular cancer therapeutics, 14(10), 2249-2259.

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HUVEC Viability and Apoptosis Assay

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HUVEC viability was assayed by adding Cell Counting Kit-8 (CCK-8, CK04, Dojindo, Rockville, MD) reagent and incubation for 4 h. HUVECs stimulated with serum-free medium (SFM) and 10% FBS were used as the controls. To evaluate EC apoptosis, HUVECs were seeded at 4 × 10⁴ cells and stimulated with 20% dHL-60 supernatant, which had been stimulated with uremic or normal serum for 20 h. Next, cells were washed with PBS and trypsinized. Detached cells were centrifuged, washed in PBS, stained using an Annexin-V-FLUOS Staining Kit (11 858 777 001, Roche), and 1 × 10⁴ cells were subjected to flow cytometry (CytoFLEX, Beckman Coulter).

Lee H.W., Nizet V., An J.N., Lee H.S., Song Y.R., Kim S.G, & Kim J.K. (2021). Uremic serum damages endothelium by provoking excessive neutrophil extracellular trap formation. Scientific Reports, 11, 21439.

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Stem Cell Antigen-1 Profiling

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Lung cells were labeled with an allophycocyanin‐conjugated anti‐(mouse stem cell antigen‐1) (Sca‐1) IgG (BD Pharmingen, San Jose, CA, USA), Annexin V and propidium iodide (PI; Annexin V‐FLUOS Staining Kit; Roche Diagnostics), and analyzed using a FACSCalibur (BD Biosciences).

Suzuki T., Ota C., Fujino N., Tando Y., Suzuki S., Yamada M., Kondo T., Okada Y, & Kubo H. (2019). Improving the viability of tissue‐resident stem cells using an organ‐preservation solution. FEBS Open Bio, 9(12), 2093-2104.

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Quantifying Apoptosis by Flow Cytometry

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Apoptotic cells were quantified by Propidium iodide (PI) and Annexin V-FITC double staining, using an Annexin-V-FLUOS Staining Kit, Roche Diagnostics according to manufacturer’s protocols. Treated and untreated cells were stained with PI and FITC according to the manufacturer protocol. The distribution of apoptotic cells was quantified by flow cytometer. A total 10,000 events were acquired.

Panja S., Ghate N.B, & Mandal N. (2016). A microalga, Euglena tuba induces apoptosis and suppresses metastasis in human lung and breast carcinoma cells through ROS-mediated regulation of MAPKs. Cancer Cell International, 16, 51.

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Apoptosis Quantification via Annexin V-FITC

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Phosphatidyl serine (PS), a phospholipid, is normally localized on the inner surface of the lipid bilayer of the plasma membrane. Externalization of PS on the other side of plasmatic membrane can be detected by the Annexin V-FITC conjugate. Annexin V staining therefore acts as a marker of programmed cell death. For apoptosis detection, floating and adherent HeLa cells (1 × 10⁶) were harvested 24, 48 and 72 h after GA treatment (c = 150 μM). NAC/GA experimental groups were pre-treated with N-Acetyl-L-cysteine (NAC c = 2 mM) for 1 h before GA was added. Complete cell population was washed in PBS and stained using Annexin-V-FLUOS Staining Kit (Roche Diagnostics, Mannheim, Germany) for 15 min at room temperature in the dark followed by incubation with propidium iodide (PI) and analyses by flow cytometer (BD FACSCalibur).

Goga M., Kello M., Vilkova M., Petrova K., Backor M., Adlassnig W, & Lang I. (2019). Oxidative stress mediated by gyrophoric acid from the lichen Umbilicaria hirsuta affected apoptosis and stress/survival pathways in HeLa cells. BMC Complementary and Alternative Medicine, 19, 221.

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Annexin-V Apoptosis Assay by Flow Cytometry

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Cells were stained with annexin-V–fluorescein isothiocyanate and propidium iodide using the annexin-V–FLUOS Staining Kit (Roche Applied Science, Penzberg, Germany). Samples were analyzed on the BD FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA) using negative and single-color controls to adjust compensation.

Liu S., Walker S.R., Nelson E.A., Cerulli R., Xiang M., Toniolo P.A., Qi J., Stone R.M., Wadleigh M., Bradner J.E, & Frank D.A. (2014). Targeting STAT5 in Hematological Malignancies through Inhibition of the Bromodomain and Extra-Terminal (BET) Bromodomain Protein BRD2. Molecular cancer therapeutics, 13(5), 1194-1205.

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