FITCAnnexin

FITCAnnexin refers to a fluorescent conjugate in which the protein Annexin V is labeled with the fluorophore fluorescein isothiocyanate (FITC). This conjugate is widely used to detect cell membrane changes associated with apoptosis, specifically the externalization of phosphatidylserine (PS). The binding of Annexin V to PS is calcium-dependent, making calcium-containing binding buffers essential for the assay. FITCAnnexin is commonly used in flow cytometry and fluorescence microscopy to identify apoptotic cells.

In typical experiments, cells are incubated with Annexin V-FITC in a calcium-containing binding buffer. Viable cells are negative for both Annexin V-FITC and a viability dye; early apoptotic cells are Annexin V-FITC positive and viability-dye negative; late apoptotic or necrotic cells are positive for both. Common viability dyes include propidium iodide (PI) or 7-Aminoactinomycin D (7-AAD), allowing discrimination of different cell states and stages of death.

Preparation and handling of Annexin V-FITC reagents vary by supplier, but general guidelines apply. Reagents are usually supplied as ready-to-use solutions or lyophilized powders, and must be protected from light. Storage is typically at 4°C for short-term use or at colder temperatures for long-term storage, with minimized freeze-thaw cycles. Working solutions are prepared in the supplied binding buffer that contains calcium ions necessary for PS binding. The fluorescence emitted by FITC is in the green region, roughly 515–525 nm, upon excitation with blue light.

Applications and limitations: FITCAnnexin is a standard tool for quantifying apoptosis in cell lines and primary cells, and is often paired with other assays to corroborate findings. PS exposure can occur in non-apoptotic processes as well, so results should be interpreted in context and with complementary methods. Photobleaching and spectral overlap with other green fluorophores can affect data quality.