Twenty-four hours after treatment, the percentage of apoptotic cells was assessed using a standard flow cytometry Annexin-V-FITC binding assay (BD Pharmingen). Briefly, cells were disaggregated by trypsin digestion and washed with PBS. The pellet was incubated at room temperature with 5 μg/ml Annexin V-FITC, 5 μg/ml propidium iodide (PI) and binding buffer for 15 min in the dark. Annexin V and PI fluorescence were measured using a Millipore Guava Easycyte 6 2L cytometer. According to the manufacturer's instructions, “cells that stain positive for FITC Annexin V and negative for PI are undergoing apoptosis. Cells that stain positive for both FITC Annexin V and PI are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both FITC Annexin V and PI are alive and not undergoing measurable apoptosis.” Data was analyzed using FlowJo 10.0.7 software.
Annexin 5 fitc binding assay
Manufactured by BD
Sourced in United States
The Annexin V–FITC binding assay is a laboratory technique used to detect and quantify apoptosis, or programmed cell death, in cell populations. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the outer membrane of cells undergoing apoptosis. The assay utilizes FITC-labeled Annexin V to fluorescently label and identify apoptotic cells.
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4 protocols using annexin 5 fitc binding assay
1
Apoptosis Quantification by Flow Cytometry
2
Annexin V-FITC Binding Assay
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Annexin V–FITC binding assay (BD Pharmingen, San Diego, CA, USA) was used as recommended by the manufacturer and analysed by flow cytometry (BD FACSCanto™, San Jose, California, USA). Analysis was performed with Diva software (FACS Diva, 6.1.2, San Jose, California, USA).
3
Annexin V-FITC Binding Assay
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Annexin V–FITC binding assay (BD Pharmingen, San Diego, California, USA) was used as recommended by the manufacturer and analyzed by flow cytometry (BD FACSCanto™, San Jose, California, USA). Analysis was performed with Diva software (FACS Diva, 6.1.2, San Jose, California, USA). Each experiment was performed in triplicate.
4
Annexin V-FITC Apoptosis Assay
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To determine apoptosis in treated cells, an annexin V-FITC binding assay (BD Biosciences Pharmingen, San Diego, CA) was performed following the manufacturer’s guidelines. Treated cells were washed with PBS buffer and then resuspended in the staining solution for 10 minutes before flow cytometry analysis. In brief, 2 × 10^6 cells in a 200 µL suspension were plated in a 6-well plate containing DMEM with 10% FCS. The cells were treated with the extract at IC50 concentrations for 24 hours. Afterward, the cells were harvested, washed, and resuspended in 5 µL of Annexin V solution, followed by incubation in the dark for 10 minutes. Then, 10 µL of propidium iodide (PI) solution was added to the cell suspension before analysis using the Coulter Epics Altra flow cytometer (Beckman Coulter, USA). The acquired data, expressed as a percentage of cells, were analyzed using the BD Accuri™ Flow cytometer. The lower left quadrant (Q3: Annexin-V and PI negative) represented viable cells, the lower right quadrant (Q4: Annexin-V positive) represented early apoptotic cells, the upper right quadrant (Q2: Annexin-V and PI-positive) represented late apoptotic cells, and the upper left quadrant (Q1: PI-positive) represented necrotic cells.
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