Annexin V-FITC Kit, 200 Tests, RUO

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• Annexin V kits employ recombinant annexin V labeled with biotin, fluorescein or R-phycoerythrin together with the viability dyes propidium iodide (PI) or 7-amino actinomycin D (7-AAD).	• Seed 5 × 10^3 cells/ well for melanoma cells and for breast cancer cells seed 3 × 10^3 cells/well in 96 well plates and permitted to adhere for 24 h at 37 °C in 5% CO2.

Upstream tips
• Annexin V kits employ recombinant annexin V labeled with biotin, fluorescein or R-phycoerythrin together with the viability dyes propidium iodide (PI) or 7-amino actinomycin D (7-AAD).
Protocol tips
• Seed 5 × 10^3 cells/ well for melanoma cells and for breast cancer cells seed 3 × 10^3 cells/well in 96 well plates and permitted to adhere for 24 h at 37 °C in 5% CO2.

Publication protocol

The assay was performed according to the manufacturer’s instructions. Briefly, the melanoma (5 × 103 cells/ well) and breast cancer cells (3 × 103 cells/well) were seeded in 96 well plates and permitted to adhere for 24 h at 37 °C in 5% CO2. The cells were treated with 1 mg/ mL of the water extract for 24 and 72 h. Detached and adherent cells were collected by trypsinization and transferred into 1.5 mL microcentrifuge tubes and centrifuged at 400 g for 5 min. The cell pellets were resuspended in 100 μL of fresh medium, to which 100 μL of Muse Annexin V & Dead Cell assay kit reagent was added. The content was mixed and incubated for 20 min at room temperature (RT) in darkness. The number of live, early apoptotic and late apoptotic/necrotic cells per 100 events were counted with the Muse Cell Analyser

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