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  • Title: Polyvinyl Alcohol can Stabilize FITC Conjugated Recombinant Annexin V for Apoptotic Cells Detection.
    Author: Shobeiri SS, Sankian M.
    Journal: Protein Pept Lett; 2022; 29(9):806-814. PubMed ID: 35718962.
    Abstract:
    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.
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