An Annexin V-fluorescein isothiocyanate (FITC)-binding assay (BD Biosciences, Franklin Lakes, New Jersey, USA) was used to assess cell apoptosis. LNCap cells were seeded in 6 well plates, were then treated with LY294002, rhEGF+LY294002, PD98059 or rhEGF+PD98059 for 24 h. Cells were washed with PBS and were stained with PI and Annexin V-FITC in each tube for 15 min, after which 1 × Binding Buffer was applied to each tube, analyzed with a BD FACS Calibur system (Beckman Coulter, CA, USA).
Annexin 5 fluorescein isothiocyanate fitc binding assay
Manufactured by BD
Sourced in United States
The Annexin V-fluorescein isothiocyanate (FITC)-binding assay is a laboratory technique used to detect and quantify the presence of phosphatidylserine (PS) on the outer membrane of cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS. When cells undergo apoptosis (programmed cell death), PS is translocated from the inner to the outer leaflet of the plasma membrane, which can be detected using the Annexin V-FITC assay.
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2 protocols using annexin 5 fluorescein isothiocyanate fitc binding assay
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Annexin V-FITC Apoptosis Assay
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Annexin V-FITC Apoptosis Assay
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Twenty-four hours after PDT, the percentage of apoptotic cells was assessed using a standard flow cytometry Annexin Vfluorescein isothiocyanate (FITC) binding assay (BD Pharmingen) according to the manufacturer's instructions. Briefly, spheroids were disaggregated by trypsin digestion and washed with PBS. The pellet was incubated on ice with 5 μg/mL Annexin V, 5 μg/mL propidium iodide (PI), and binding buffer for 15 min in the dark. Annexin V and PI fluorescence were measured using a FACSCanto II flow cytometer (BD Biosciences). Early apoptotic cells are Annexin V-positive and PI-negative (Annexin V-FITC + / PI -), whereas late (end-stage) apoptotic cells are Annexin V/PI-double-positive (Annexin V-FITC + /PI + ). Necrotic cells are Annexin V-negative and PI-positive (Annexin V-FITC -/PI + ) [21] . Data was analyzed using FlowJo 10.0.7 software (Tree Star Inc, Ashland, USA).
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