EXPERIMENT 1: APOPTOSIS STAINING

 FITC Annexin V Apoptosis Detection Kit with 7-AAD

FITC Annexin V Apoptosis Detection Kit with 7-AAD: It has benn specifically designed
fort he 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.

Phosphatidylserine: The
exposure of phosphatidylserine
(PS) on the outer plasma
membrane has long been
considered a unique feature of
apoptotic cells. Together with
other “eat me” signals, it
enables the recognition and
phagocytosis of dying cells
(efferocytosis), helping to explain the immunologically-silent natüre of apoptosis.

Annexin A5 or Annexin V is a cellular protein in the annexin group. While Annexin A5 is

located on the outside of the plasma
membrane in flow cytometry, it is often
used because of its ability to bind to the
apoptosis marker phosphatidylserine.

Necrotic Cell: Necros is the death of cells

in your body. Necros ca occur because of
injuries, infections or diseases. Lack of
blood flow to your tissues and extreme
environmental conditions can also cause
the necros.
Staining Procedure

1. The cells are washed twice with cold Biolegend Cell Staining Buffer and the cells
are resuspended in Annexin V Binding Buffer at a concentration of 0.25-1.00 X
107 consantration.
2. 100 µL cell suspension are placed in a 5 ml test tube.
3. 5 µL FITC Annexin V are added.
4. 5 µL 7-AAD Viability Staining solution is added.
5. The cells are gently vortexed and are incubated at room temperature for 15
minutes in the dark.
6. 400 µL Annexin V Binding Buffer are added to each the tube and they are
analized with the current machine setting of the flow cytometer.