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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Published on: April 24, 2011
Polyvinyl Alcohol can Stabilize FITC Conjugated Recombinant Annexin V for Apoptotic Cells Detection
Saeideh Sadat Shobeiri1, Mojtaba Sankian1
1Immunology Research Center, Faculty of medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Background:
Annexin V, a member of calcium-dependent phospholipid-binding proteins, selectively binds to the exposed phosphatidylserine, which can be used for in vitro apoptosis detection. Simultaneous staining of cells with annexin V-fluorescein isothiocyanate (FITC) and the non-vital dye propidium iodide (PI) enables the detection of apoptotic and necrotic cells.
Objective:
Our study aimed to express, purify, and stabilize the recombinant annexin V.
Methods:
The recombinant annexin V was cloned and expressed in E. coli bacteria and was purified using Ni-IDA resin. The FITC conjugation was performed, and apoptosis detection of HaCaT cells by FITC-labeled annexin V was evaluated by flow cytometry. Then, the stability of FITC-labeled annexin in various conditions, including polyvinyl alcohol (PVA), glycerol, and trehalose, was evaluated.
Results:
The results showed that annexin V was appropriately expressed and purified. After FITC conjugation, it could perfectly detect the cell death of HaCat cells in different apoptosis percentages. FITC-labeled annexin had more stability with PVA than glycerol and trehalose.
Conclusion:
It seems that PVA has an acceptable effect on FITC-labeled annexin V stability in concentrations lower than 1 mg mL-1 without interfering with fluorescent intensity.
