For evaluation of fisetin, quercetin, kaempferol, and their CD complexes effects on B164A5 melanoma cells, these cells were seeded on 6-well culture plates at a cellular density of 10,000 cells/cm 2 . The tested substances were added in culture media at a concentration of 100 µM and left to interact for 72 hours. Annexin V-FITC (Miltenyi Biotec, Gladbach, Germany) was used in cell death flow cytometric studies (apoptosis) combined with propidium iodide staining solution (BD Biosciences, San Jose, CA, USA) following the manufacturer's protocol. Briefly, 10 6 cells were washed in 1 × Annexin V Binding Buffer (BD Pharmigen), centrifuged at 300 g for 10 min, resuspended in the same solution and incubated with 10 µL of Annexin V-FITC for 15 min in the dark. After washing the cells with 1 mL specific binding buffer and centrifugation, the cell pellet was resuspended in 500 µL binding buffer, and 1 µg/mL of PI solution was added immediately prior to analysis by flow cytometry. Acquisition of data was performed with the Cell Quest Pro software (Becton-Dickinson) on a 2 lasercapable flow cytometer (FACSCalibur; BD Biosciences), and data analyses used Flowing Software 2.5.
Annexin 5 fitc
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
Annexin V-FITC is a fluorescently labeled protein that binds to phosphatidylserine, a phospholipid that is exposed on the surface of cells undergoing apoptosis. It is a tool used in flow cytometry and other cell-based assays to detect and quantify apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (6 mentions)
Facscalibur, by BD (4 mentions)
Annexin 5 fitc, by BD (4 mentions)
Facscanto 2 flow cytometer, by BD (4 mentions)
Propidium iodide, by Miltenyi Biotec (4 mentions)
Annexin 5 binding buffer, by BD (3 mentions)
Propidium iodide staining solution, by BD (3 mentions)
Annexin 5 binding buffer, by Miltenyi Biotec (3 mentions)
Facscanto 2, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
7 aad staining solution, by BD (2 mentions)
Mouse anti f4 80 pe clone rea126, by Miltenyi Biotec (2 mentions)
Rnase a, by Qiagen (2 mentions)
1 mg ml stock solution, by Merck Group (2 mentions)
Propidium iodide solution, by Miltenyi Biotec (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Cellquest pro software, by BD (1 mentions)
Facsdiva version 6, by BD (1 mentions)
Anti cd44 pe, by BD (1 mentions)
Canto cytometer, by BD (1 mentions)
51 protocols using annexin 5 fitc
1
Evaluating Flavonoid Effects on Melanoma Cells
2
Evaluating Cell Response to Targeted Therapies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines and sub-factions were treated with Cetuximab and Erlotinib before double staining with Annexin V-FITC and DAPI, or staining with anti-ESA-APC (Miltenyi Biotec), anti-CD44-PE (BD Pharmingen), Annexin V-FITC (BD Pharmingen) and DAPI. Samples were examined on a Canto Cytometer (BD Bioscience) and analyzed with the FACS Diva version 6.1.1 (BD Biosciences) software.
3
Cell death and cell cycle analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Changes in Ψm (ΔΨm) were measured by DiOC6 (Molecular Probes, Dreieich, Germany) staining [11 (link)]. For annexin-V/PI staining, cells were washed in PBS, resuspended in 50 µL 1x annexin-V binding buffer (10× stock: 100 mM HEPES, 1,4 M NaCl, 25 mM CaCl2, 1% BSA, pH 7,4). Staining was performed with 2.5 µL annexin V-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) at room temperature for 15 min in the dark. Before measurement, 10 µL PI (stock solution: 50 µg/mL) were diluted in 430 µL annexin binding buffer and added to the cell suspension. Viable cells are annexin-V-/PI-negative, early apoptotic cells are annexin-V-positive/PI-negative; late apoptotic or necrotic cells are annexin-V-positive/PI positive [11 (link),22 (link)]. For cell cycle analysis, cells were washed with PBS, resuspended in 100 µL PBS, and fixed with 2 mL ice-cold 80% ethanol for 1 h up to 1 week at −20 °C. After centrifugation, ethanol was removed, and cells were incubated in 1 µL RNase-A (1 µg/mL) (Sigma-Aldrich, Taufkirchen, Germany) per 333 µL PBS (1 h, room temperature). Cells were stained with 167 µL propidium iodide (PI; Sigma-Aldrich, Taufkirchen, Germany; stock solution: 50 µg/mL). Samples were measured on a FACS Canto II flow cytometer with FACSDiva 7.0 software (BD-Biosciences, Heidelberg Germany; FITC-channel).
4
Oxidative Stress-Induced Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were seeded in 12 well plates at the density of 1 × 106 cells per ml per well. After incubation for 24 h at 37 °C with 50 μM of etoposide (Sigma™) to induce oxidative stress, cells were harvested and washed twice with PBS-EDTA. Apoptotic cells were identified with the annexin-V-FITC apoptosis detection kit from BD Pharmingen kit (556547) following the manufacturer’s instructions. PBMCs were immunostained for CD45, CD19 (eBioscience) or CD3, and CD4 (Miltenyi) prior to annexin-V-FITC/IP staining. The apoptotic cells were analyzed by flow cytometry, as described above.
5
Apoptosis Induction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1,10-Phenanthroline-5,6-dione, lactacystin, triton X-100, and propidium iodide (PI) were from Sigma-Aldrich Chemie GmbH, Munich, Germany; annexin V-FITC was from Miltenyi Biotec, Bergisch Gladbach, Germany; chloroquine was from Enzo Life Sciences GmbH, Lörrach, Germany; Z-VAD-FMK and the HSP70 inhibitor JG-98 were from Selleck Chemicals, Munich, Germany; dithiothreitol was from PanReac AppliChem, Darmstadt, Germany, formalin (37%) and bovine milk powder were purchased from Carl Roth, Karlsruhe, Germany.
6
Quantifying Apoptosis via Annexin V-FITC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptosis were quantified using Annexin V-FITC (Miltenyi, 130-092-052) to detect externalized phosphatidylserine and PI (Miltenyi, 130-092-052) to detect plasma membrane disruption. Tumor cells were firstly pretreated with DDP (10 mg/mL) for 24 h, and then assessed with Annexin V and PI in binding buffer for 15 min in the dark. Cells were collected using flow cytometer (BD Calibur) and the data were analyzed using FlowJo software.
7
Annexin V-FITC and Propidium Iodide Assay for Apoptosis and Necrosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the determination of apoptosis and necrosis the annexin V/propidium iodide (AV/PI) assay coupled with flow cytometry analysis was used. In brief, for harvest cells in the supernatant were collected in a 15 mL tube, samples were washed twice with PBS and detached with trypsin/EDTA solution. They were washed twice in PBS and 50 μL 1× binding buffer and 2.5 μL Annexin V/FITC (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added to each sample. Following 15 min incubation in the dark on ice, 430 μL 1x binding buffer and 1 µg/mL PI (Sigma-Aldrich, Steinheim, Germany) were added to the cells. Data acquisition was done by a FACS Canto II flow cytometer (Becton Dickinson GmbH, Heidelberg, Germany). Annexin V positive cells were classified as apoptotic while annexin V and PI double-positive cells were classified as necrotic/late-apoptotic. The data were analysed using the BD FACSDiva software. A representative plot of control and treated cells is shown in Supplementary Materials, Figure S1 .
8
Annexin-V-FITC Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jurkat cells were treated for 5 h with 80 μM BSB, washed with PBS, and incubated with Annexin-V-FITC (Miltenyi Biotec, Bergisch Gladbach, DE) for 15 min. TO-PRO-3 (Invitrogen) was added immediately before the flow cytometry acquisition.
9
Quantifying Endothelial Cell Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The apoptosis of the EPCs was evaluated by flow cytometry. The EPCs were fluorescently labeled by the addition of 500 µl of binding buffer, 5 µl of Annexin V-FITC and 5 µl of propidium iodide (Miltenyi Biotec, Bergisch Gladbach, Germany). Following incubation in the dark at room temperature for 15 min, the cells were subjected to flow cytometric analysis. A minimum of 10,000 cells in the gated region was analyzed using a BD FACSCalibur Flow Cytometer (Becton-Dickinson). The esults were presented by the percentage of total cells.
10
Apoptosis Evaluation by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effects of changes of COPB2 expression on apoptosis were examined by flow cytometry. Cells at the log phase were seeded in a medium at a density of 5×105 cells per well, and incubated in a 6-well plate for 24 h. The cells were then washed in phosphate buffered saline (PBS), centrifuged at 1,000 r/min for 5 min. Five µL Annexin V-FITC (130-093-060, Miltenyi, Germany) was added to the cells, gently mixed and incubated for 15 min, the supernatant was discarded, and the cells were added with 10 µL propidium iodide (PI, JBS-LI-001, ENZO, USA). Finally, the apoptosis of cells were analyzed by flow cytometry (FCM, 322457, Bio-Rad, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!