Cell death was analyzed by flow cytometry as described previously [25 (link)]. For Annexin V/7-AAD staining, cells at 24 h after treatment of each flavonoid were washed twice with PBS and stained with FITC-conjugated Annexin V antibody (BD Bioscience, # 556,419) and 7-AAD (BD Bioscience, #559,925) for an additional 45–60 min at room temperature in the dark. Cells stained with Annexin V/7-AAD were analyzed by FACS Calibur or FACS Lyric (BD Bioscience). For the bright field images captured, light channel of optical microscope (Olympus, CKX-41) or JuLI-stage (NanoEntek, Korea) was used in accordance with the manufacture’s protocol. JuLI-STAT was used for analysis of the data from JuLI-stage (NanoEntek, Korea). The activity of caspase-3 was analyzed by colorimetric active caspase-3 assay kit (Sigma-Aldrich, #CASP3C) in accordance with the manufacturer's protocol.
Fitc conjugated annexin 5 antibody
Manufactured by BD
Sourced in United States
The FITC-conjugated annexin V antibody is a fluorescent probe used to detect and quantify apoptotic cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the surface of apoptotic cells. The FITC (fluorescein isothiocyanate) conjugation allows for the visualization and analysis of apoptotic cells by flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by BD (5 mentions)
7 aad, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Juli stage, by NanoEnTek (3 mentions)
Ckx 41, by NanoEnTek (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Facslyric, by BD (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Juli stat, by NanoEnTek (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by BD (1 mentions)
Casp 3 c, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facs caliber flow cytometer, by BD (1 mentions)
In situ cell death kit, by Roche (1 mentions)
Annexin v 7 aad, by BD (1 mentions)
16 protocols using fitc conjugated annexin 5 antibody
1
Apoptosis Quantification by Flow Cytometry
2
Annexin V-based Apoptosis Detection
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Cells were seeded and treated as described for the cell cycle analysis. Cells and washings were pelleted by centrifugation at 600 g , washed twice with PBS and resuspended in 600 μl Annexin V-binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl and 2.5 mM CaCl2). A 150-μl aliquot of the cell suspension was stained with 5 μl of propidium iodide (Invitrogen, Carlsbad, CA, USA) at 50 g l−1 and 4 μl of FITC-conjugated Annexin V antibody (BD Biosciences, San Jose, CA, USA). The apoptotic status of the cells was analysed using a FACSCalibur flow cytometer (Becton Dickinson). Cells undergoing apoptosis were defined as positive for Annexin V and negative for propidium iodide staining.
3
Flow Cytometry Analysis of Flavonoid-Induced Cell Death
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Cell death was analyzed by flow-cytometry as described previously [25 (link)]. Regarding Annexin V/7-AAD staining, cells at 24 h after treatment of each flavonoid were washed twice with PBS and stained with FITC conjugated Annexin V antibody (BD Bioscience, Franklin Lakes, NJ, USA, #556419) and 7-AAD (BD Bioscience, #559925) for an additional 45–60 min at room temperature in the dark. Cells stained with Annexin V/7-AAD were analyzed by FACS Calibur or FACS Lyric (BD Bioscience). Concerning all of the bright field images captured, a Light channel optical microscope (Olympus, Tokyo, Japan, CKX-41) or JULI-stage (NanoEntek, Seoul, Korea) was used in accordance with the manufacture’s protocol. The activity of caspase-3 was analyzed using a colorimetric active caspase-3 assay kit (Sigma–Aldrich, #CASP3C) according to the manufacture’s protocol.
4
Apoptosis Quantification by Flow Cytometry
5
Apoptosis Assay in HASM Cells
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HASM cells were seeded in 4-well chamber slides (Lab-Tek, Nunc) and serum-starved for 48 hours. Cells were left untreated or treated with vehicle, Af (5 µg ml−1) for 48 hours or with staurosporine (1 µM) for 6 hours. Cells were then stained with FITC-conjugated Annexin V-antibody and propidium iodide (PI) as described by the manufacturer (BD Biosciences). Images were obtained using a Leica DMI4000 Fluorescence microscope equipped with Retiga2000R camera (QImaging, Canada) and analyzed using Image-pro Plus software (Media Cybernetics).
6
Apoptosis Assay for Immune Cells
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The mononuclear cells isolated from spleen (1 × 105 cells/sample) were resuspended in 1× Binding buffer (10× Binding buffer contained 0.1 M HEPES, 1.4 M NaCl, 25 mM CaCl2 in distilled water, pH = 7.4) and labeled with FITC-conjugated Annexin V antibody (BD Biosciences) and Propidium iodide (PI) (50 μg/ml) (Sigma-Aldrich) for 15 min on RT. For the assessment of apoptosis of different immune cells, additional staining with fluorochrome-conjugated anti-mouse CD11c, CD19, F4/80 and CD3 antibodies (BD Biosciences/Miltenyi Biotec GmbH) was performed. Further flow cytometric analysis was conducted using FACSCalibur Flow Cytometer (BD Biosciences). The data were analyzed with FlowJo (Tree Star).
7
Apoptosis Detection in H9c2 Cells
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Treated H9c2 Cells were harvested and incubated with FITC-conjugated annexin V antibody (BD Biosciences) and PI at room temperature for 20 min before they were analyzed using flow cytometry. The results were further analyzed using WinMDI software.
8
Apoptosis Quantification by Flow Cytometry
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The cells were treated with rJHP0290 (100 ng/ml) and/or CPT (10 μM) (Sigma-Aldrich), as indicated in the figure legends. The cells were stained with a FITC-conjugated Annexin V antibody (BD Biosciences) and PI (BD Biosciences) according to the manufacturer’s instructions. Briefly, 1x105 cells in 100 μl of Annexin binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) were mixed with 1 μl of FITC-conjugated Annexin V antibody. The mixture was incubated for 15 min at room temperature in the dark. PI (2.5 μl) was added to the cell suspension immediately prior to analysis. The relative number of Annexin V-positive and/or PI-positive cells was determined using flow cytometry. FlowJo software was used for the data analysis.
9
Apoptosis Quantification in Keloid Fibroblasts
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Keloid fibroblast cells (1.0 × 106 cells) were harvested, washed, and fixed in ice-cold ethanol. Then, cells were re-suspended in the staining solution containing 5 µL of FITC-conjugated annexin V antibody and 5 µL of propidium iodide (BD Bioscience, San Jose, CA, USA). After incubation in the dark, the apoptotic cells were detected on a flow cytometer (BD Bioscience, San Jose, CA, USA).
10
Assessing Cell Proliferation and Apoptosis
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A total of 5 × 104 ES49 cells or 1 × 105 EWS cells were seeded in 12‐well plates. HA‐tagged Trib1 and Nrg1 cDNAs were subcloned into pCDNA3.1 and transfected into ES49 or EWS cells. Cell proliferation was determined by measuring cell numbers 48 and/or 72 hours after siRNA treatment using the Countess cell number counter (Invitrogen, La Jolla, CA, USA). For apoptosis and cell cycle assays, siRNA‐treated cells were cultured with an FBS‐depleted medium (0.5% FBS) for 48 hours. To detect apoptotic cells, cells were stained for Annexin‐V using an FITC‐conjugated Annexin‐V antibody (BD Biosciences, Franklin Lakes, NJ, USA). DNA synthesis was assessed by 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation using the Click‐iT EdU Alexa Fluor 647 detection kit (Molecular Probes, Eugene, OR, USA). Processed cells were examined by FACSCalibur Flow Cytometer (BD Biosciences). FOXQ1 was detected by immunoblotting.
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