How does annexin V detect apoptosis?
Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane.
Is annexin V staining an apoptosis assay?
As a new method for detecting apoptosis, the Annexin V binding assay is based on the measurement of the loss of plasma membrane asymmetry.
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.
How do you detect apoptosis?
A common method capable of assaying the state of the cell chromatin is the DNA ladder assay. Breaks in chromatin result in the formation of DNA fragments, which can be detected by several methods. When detected by gel electrophoresis, the ladder formation of the fragmented DNA can be used to characterize apoptosis.
Why does propidium iodide staining indicate apoptosis?
Propidium Iodide Propidium Iodide (PI) is a fluorescent DNA-binding dye. It freely penetrates cell membranes of dead or dying cells, but is excluded from viable cells. These properties favor the use of PI for evaluation of (apoptotic) cell death or for cell cycle analysis.
What are the markers of apoptosis?
The markers most commonly used for in vitro detection of apoptosis include caspase-3/7 activity and phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane.
Does propidium iodide detect apoptosis?
Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models.
Is annexin V staining a marker for apoptosis?
While cells positive for both probes may indicate late stage apoptosis, annexin V staining in this population may be due to inner leaflet PS binding and therefore may not be reliably deemed apoptotic. In this combination, cells that are only annexin V-positive and therefore have intact plasma membranes, are demonstrably apoptotic.
What is the FITC annexin V apoptosis detection kit?
BioLegend’s FITC Annexin V Apoptosis Detection Kit with PI has been specifically designed for the identification of apoptotic and necrotic cells. Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner.
How can I control the specificity of annexin V-FITC staining?
An additional control that may be performed includes preincubation of cell samples with recombinant unconjugated Annexin V, which is included as part of the Annexin V-FITC Apoptosis Detection Kit II (Cat. No 556570). This serves to block Annexin V-FITC binding sites and thus demonstrates the specificity of Annexin V-FITC staining.
How do I bind annexin V-biotin to human cells?
Gently mix the cells and incubate for 15 min at RT in the dark. *For Annexin V-Biotin samples only: After 15 min incubation, wash once with 1 ml of 1X Binding Buffer. Dilute 0.5 µg Streptavidin-Fluorescein Isothiocynate (SAv-FITC, Cat. No. 554060) into 100 µl of 1X Binding Buffer and add to the cell pellet. Gently mix the cells.