Annexin 5 fitc

Manufactured by Southern Biotech

Sourced in United States

Annexin V-FITC is a fluorescent dye-labeled protein that binds to phosphatidylserine, a cellular membrane component. It is commonly used in flow cytometry and fluorescence microscopy to detect and quantify apoptotic cells.

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Lab products found in correlation

Propidium iodide, by Merck Group (4 mentions) Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions) Sytox green, by Thermo Fisher Scientific (3 mentions) Mitotracker red, by Thermo Fisher Scientific (3 mentions) Guava easycyte plus flow cytometer, by Merck Group (2 mentions) Celigo s imaging cytometer, by Revvity (2 mentions) Viastain live caspase 3 7 detection kit, by Revvity (2 mentions) Cellquest software, by BD (2 mentions) Facscalibur, by BD (2 mentions) Lsr 2, by BD (1 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Dcfh da, by Merck Group (1 mentions) Accuri c6, by BD (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Annexin 5 fitc, by BD (1 mentions) Guava flow cytometry system, by Merck Group (1 mentions) Anti cd14 pe, by Thermo Fisher Scientific (1 mentions) Guava easycyte plus, by Merck Group (1 mentions) Lysotracker deep red, by Thermo Fisher Scientific (1 mentions) Sytox blue, by Thermo Fisher Scientific (1 mentions)

10 protocols using annexin 5 fitc

Annexin V and Caspase 3/7 Apoptosis Assay

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For annexin V staining, cells were trypsinized and resuspended in binding buffer at 72 h post-transfection and incubated with Annexin-V-FITC (Southern Biotech) and propidium iodide (Sigma–Aldrich) for 15 min at room temperature. Samples were analyzed using a Guava EasyCyte Plus flow cytometer (Guava Technologies).
The kinetic apoptosis assay for caspase 3/7 activity was performed according to the manufacturer’s instructions (CS1-V0002(3)-1, ViaStainTM Live Caspase 3/7 Detection Kit, Nexcelom). Scanning was performed 24, 48, and 72 h post-transfection using a Celigo^® S imaging cytometer (Nexcelom Bioscience LLC). For detailed protocols, please refer to the Supplementary Data.

Wegwitz F., Prokakis E., Pejkovska A., Kosinsky R.L., Glatzel M., Pantel K., Wikman H, & Johnsen S.A. (2020). The histone H2B ubiquitin ligase RNF40 is required for HER2-driven mammary tumorigenesis. Cell Death & Disease, 11(10), 873.

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Apoptosis Analysis by Flow Cytometry

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Seventy-two hours post transfection, adherent, and floating cells were collected, washed twice with PBS, and resuspended in binding buffer (10 mM HEPES, pH 7.4; 140 mM NaCl; 25 mM CaCl₂) at a concentration of 10⁵ cells in 100 µl. 5 µl Annexin V-FITC (Southern Biotech) and 1 µl propidium iodide (Sigma Aldrich) were added per sample, gently mixed, and incubated for 15 min at room temperature in the dark. 400 µl binding buffer was subsequently added to each sample. Analysis of the Annexin V staining was performed using a Guava EasyCyte Plus flow cytometer (Guava Technologies).

Prokakis E., Dyas A., Grün R., Fritzsche S., Bedi U., Kazerouni Z.B., Kosinsky R.L., Johnsen S.A, & Wegwitz F. (2021). USP22 promotes HER2-driven mammary carcinoma aggressiveness by suppressing the unfolded protein response. Oncogene, 40(23), 4004-4018.

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Leukocyte Apoptosis Assay

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Tumor-infiltrating leukocytes isolated as described above were first stained with antibodies against surface markers in MACS buffer. After washing, cells were resuspended in Annexin V binding buffer and stained with Annexin V FITC (SouthernBiotech, cat#10010-02). Alternatively, cells in MACS buffer were incubated with CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific, cat#C10740) for 25 min at 37 °C, and then incubated with SYTOX AADvanced dead cell stain solution for 5 min at 37 °C. Stained samples were analyzed with a BD LSR II for Annexin V positive or Caspase-3/7 positive cells.

Liu Y., Debo B., Li M., Shi Z., Sheng W, & Shi Y. (2021). LSD1 inhibition sustains T cell invigoration with a durable response to PD-1 blockade. Nature Communications, 12, 6831.

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Multiparameter Flow Cytometry Protocols

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A BD FACSCalibur or Accuri C6 flow cytometer was used. Experiments were performed at least twice, with 3-6 replicates per condition, and 10,000- 50,000 cells collected per sample, analyzed via the FLOJO software package. Cell cycle: Cells were fixed with cold 70% ethanol overnight, washed in PBS/5% FBS, stained in PBS/5% FBS with 10μg/ml Propidium Iodide (PI; Molecular Probes), and 100 μg/ml of RNase A. Annexin V staining: cells were stained with Annexin V- FITC (Southern Biotechnology 10038-02) in Annexin staining buffer. PI (Molecular Probes, P1304MP) was added just before analysis per manufacturer's instructions. ROS Detection: cultures were incubated for 15-30 min with 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma) washed (PBS), harvested (trypsin), and analyzed.

Cobler L., Zhang H., Suri P., Park C, & Timmerman L.A. (2018). xCT inhibition sensitizes tumors to γ-radiation via glutathione reduction. Oncotarget, 9(64), 32280-32297.

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Apoptosis Quantification in Melanoma Cells

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Double staining for Annexin-V–FITC binding and DNA using PI was carried out in B16F10 and SKMEL-28 cells exposed to ethanol or DDA. Cells were washed in PBS and resuspended in the binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, 0.1% bovine serum albumin, pH 7.4). Cell suspensions were then incubated on ice with Annexin-V-FITC (Southern Biotech, Birmingham, AL, USA). After 15 min, an additional 380 μl of the binding buffer was added, followed by 0.5 mg/ml PI immediately before analysis with a BD Facscalibur flow cytometer (BD Biosciences). The percentage of Annexin-V–FITC/PI-positive cells was determined using CellQuest software (BD Biosciences).

Segala G., David M., de Medina P., Poirot M.C., Serhan N., Vergez F., Mougel A., Saland E., Carayon K., Leignadier J., Caron N., Voisin M., Cherier J., Ligat L., Lopez F., Noguer E., Rives A., Payré B., Saati T.A., Lamaziere A., Despres G., Lobaccaro J.M., Baron S., Demur C., de Toni F., Larrue C., Boutzen H., Thomas F., Sarry J.E., Tosolini M., Picard D., Record M., Récher C., Poirot M, & Silvente-Poirot S. (2017). Dendrogenin A drives LXR to trigger lethal autophagy in cancers. Nature Communications, 8, 1903.

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Apoptosis Assay with Annexin V-FITC

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Floating cells were collected and adherent cells were trypsinized, filtered, and washed with ice-cold PBS. After centrifugation, 100,000 cells were resuspended in 100 μl binding buffer (0.01 M HEPES pH 7.4, 0.14 M NaCl, 2.5 mM CaCl₂). Propidium iodide (Sigma Aldrich) and Annexin V-FITC (Southern Biotech) were added (1:100) and incubated at room temperature for 15 min. Four hundred microliters of binding buffer was added, and apoptosis analyses were performed using the Guava flow cytometry system (Millipore) using the same gate settings for each cell line. The experiment was performed with three biological replicates per condition, each with two technical replicates in three independent experiments.

Schneider D., Chua R.L., Molitor N., Hamdan F.H., Rettenmeier E.M., Prokakis E., Mishra V.K., Kari V., Wegwitz F., Johnsen S.A, & Kosinsky R.L. (2019). The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells. Clinical Epigenetics, 11, 98.

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Monocyte Apoptosis Analysis by Flow Cytometry

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Apoptosis of CD14⁺ monocyte was assessed using Southern Biotech Apo Screen Annexin V apoptosis detection kit (Annexin V-FITC, 7-AAD solution and Annexin V binding buffer). This assay involves staining peripheral blood mononuclear cells with Annexin V-FITC (a phospholipid-binding protein binding to disrupted cell membranes) in combination with 7-AAD (a vital dye binding to DNA penetrating into apoptotic cells). Fluorescence-activated cell sorting (FACS) analysis of CD14⁺ monocytes that are in early (annexin V⁺/7-AAD⁻) or late (annexin V⁺/7-AAD⁺) apoptotic phase was performed. The percentage of apoptotic CD14⁺ monocytes (out of total circulating CD14⁺ cells) was assessed by staining peripheral blood mononuclear cells for 3 color FACS analysis employing anti-CD14-PE (eBioscience), Annexin V-FITC and 7-AAD (SouthernBiotech). The cells were then washed again with PBS and re-suspended in 100 µL Annexin V-FITC binding buffer and incubated with 5 µL of Annexin V-FITC for 15 min at room temperature. Another 200 µL of binding buffer and 5 µL of 7-AAD solution were added without washing, and 100,000 cells were acquired by flow cytometry (FACS Calibur, Becton Dickinson) and analyzed by CellQuest software (BD Bioscience). All analyses and readings were made by researchers who were blinded to the study questions.

Shimoni S., Meledin V., Bar I., Fabricant J., Gandelman G, & George J. (2016). Circulating CD14+ monocytes in patients with aortic stenosis. Journal of Geriatric Cardiology : JGC, 13(1), 81-87.

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Annexin V and Caspase 3/7 Apoptosis Assays

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For annexin V staining, cells were tryprinized and resuspended in binding buffer at 72 hours post-transfection and incubated with Annexin V-FITC (Southern Biotech) and propidium iodide (Sigma Aldrich) for 15 min at room temperature. Samples were analyzed using a Guava EasyCyte Plus flow cytometer (Guava Technologies).
The kinetic apoptosis assay using caspase 3/7 was performed according to the manufacturer's instructions (CS1-V0002(3)-1, ViaStainTM Live Caspase 3/7 Detection Kit, Nexcelom). Scanning was performed at time points 24, 48 and 72 hours post transfection using a Celigo® S imaging cytometer (Nexcelom Bioscience LLC). For detailed protocols, please refer to the Supplementary Data.

Wegwitz F., Prokakis E., Pejkovska A., Kosinsky R.L., Glatzel M., Pantel K., Wikman H., & Johnsen S.A. (2020). RNF40-dependent epigenetic regulation of actin cytoskeletal dynamics is required for HER2-driven mammary tumorigenesis.

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Apoptosis Induction and Protection Assay

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Suspension cells (U-937, SKW 6.4, or Jurkat cell lines) were plated into a 24 well plate (0.5 × 10⁶ cells/mL). The drugs of interest were added from DMSO stocks (1% v/v final DMSO). The cells were incubated for the appropriate time after which they were centrifuged and resuspended in annexin V binding buffer containing 1 μg/mL propidium iodide (PI) and 100X dilution of FITC-annexin V (Southern Biotechnology). The samples were analyzed by cell flow cytometry on a Benton Dickinson LSRII flow cytometer. For protection assays, U-937 cells were pretreated with the prospective protective agents for 2 hours at concentrations listed in Detailed Experimental Procedures, after which the cells were then co-treated with 10 μM Raptinal for 2 hours prior to analysis.

Palchaudhuri R., Lambrecht M.J., Botham R.C., Partlow K.C., Van Ham T.J., Putt K.S., Nguyen L.T., Kim S.H., Peterson R.T., Fan T.M, & Hergenrother P.J. (2015). A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed. Cell reports, 13(9), 2027-2036.

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Fluorescent Dyes and Probes for Cell Analysis

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Invitrogen™ H33342 (H3570), propidium iodide (PI; P3566), Sytox™ blue (S11348), Sytox™ red (S34859), Sytox™ green (S7020), Sytox™ orange (S11368), Lysotracker™ deep red (L12492), Lysotracker™ red DND-99 (L7528), Mitotracker™ red (M22425), Mitotracker™ deep red FM (M22426), CM-H2-DCFDA (C6827), BODIPY™ 581/591 C11 (D3861), Cascade Blue™ dextran 3 kDa (D7132), Alexa Fluor™ 488 dextran 10 kDa (D22910), tetramethylrhodamine (TMR) dextrans 40 kDa (D1842) and 70 kDa (D1818), and Texas red labeled dextrans 3 kDa (D3328), 10 kDa (D1863), 40 kDa (D1829), and 70 kDa (D1864) were purchased from Thermo Fisher Scientific. Acridine orange (AO) (40039) and NucView^® 530 (10406) were purchased from Biotium. FITC-annexin V was purchased from Southern Biotech or FITC-annexin V apoptosis detection kit (556547) was purchased from BD Pharmingen.

Luke C.J., Markovina S., Good M., Wight I.E., Thomas B.J., Linneman J.M., Lanik W.E., Koroleva O., Coffman M.R., Miedel M.T., Gong Q., Andress A., Campos Guerrero M., Wang S., Chen L., Beatty W.L., Hausmann K.N., White F.V., Fitzpatrick J.A., Orvedahl A., Pak S.C, & Silverman G.A. (2022). Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins. Communications Biology, 5, 47.

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