What does annexin stain?
Annexin V staining is a common method for detecting apoptotic cells. Thermo Fisher Scientific offers high-quality fluorescent annexin V conjugates as standalone reagents and in a variety of kits for use in flow cytometry and for imaging suspension cells.
How do you use annexin V?
Add 5 μL of fluorochrome-conjugated Annexin V to 100 μL of the cell suspension. Incubate 10-15 minutes at room temperature. Protect from light. Add 2 mL 1X binding buffer and centrifuge at 400-600 x g for 5 minutes at room temperature.
Does PI stain apoptotic cells?
The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6.
How do you dissolve PI?
Propidium iodide (PI) is supplied as a crystalline solid. A stock solution may be made by dissolving the PI in the solvent of choice. PI is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide, which should be purged with an inert gas.
What does propidium iodide stain?
Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis, or in microscopy to visualize the nucleus and other DNA-containing organelles.
What is annexin V in apoptosis?
Annexin A5 (or annexin V) is a cellular protein in the annexin group. In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane.
How to mix annexin V with dissolved Pi?
Add 1 μL of annexin V-FITC solution and 5 μL of dissolved PI to 100 μL of the cell suspensions prepared as given in step 1. Mix gently. Keep tubes on ice and incubate for 15 minutes in the dark. Add 400 μL of ice-cold 1X binding buffer and mix gently.
How much annexin V is needed for staining?
Add 5-15 µg of purified recombinant Annexin V. The amount of purified recombinant Annexin V required to saturate binding sites may vary according to cell type, and stage of apoptosis.
What can PI staining solution be used for?
The PI Staining Solution is designed for use in two-color Annexin V flow cytometric assays. It can be used in conjunction with Annexin V conjugated to fluorescein isothiocyanate (Annexin V-FITC, Cat. No. 556419) or biotinylated Annexin V (Annexin V-Biotin, Cat. No. 556417). Suggested amounts to use are 10 µl/test (1x10e6 cells).
How does annexin V stain in early apoptosis?
These cells will stain with Annexin V but not with viability dyes, thus distinguishing cells in early apoptosis. In late stage apoptosis, the cell membrane loses integrity, thereby allowing Annexin V to also access PS in the interior of the cell.