1

EXPT NO: 7

DETECTION OF APOPTOTIC PROTEINS BY FLORESCENCE MICROSCOPY

_____________________________
AIM:
To detect the apoptotic proteins by annexins v staining method

PRINCIPLE:
Annexins are a family of calcium dependent phospholipid binding proteins that
preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is
predominantly located in the inner leaflet of the plasma membrane. Upon induction of
apoptosis, rapid alterations in the organisation of phospholipids in most cell types occurs
leading to exposure of PS on the cell surface. In vitro detection of externalized PS can be
achieved through interaction with the anticoagulant annexin V. In the presence of calcium,
rapid high affinity binding of annexin V to PS occurs. PS translocation to the cell surface
precedes nuclear breakdown, DNA fragmentation, and the appearance of most apoptosis-
associated molecules.  Annexin V conjugated to FITC bind to phosphatidylserine on the outer
surface of cells undergoing apoptosis. Fluorescence is then detected by fluorescence
microscopy. Since membrane permeabilization is also observed in necrosis, necrotic cells will
also bind Annexin V-FITC. Propidium iodide is included to help distinguish between viable,
early apoptotic, and necrotic or late apoptotic cells. Necrotic cells will bind Annexin V-FITC
and stain with propidium iodide while propidium iodide will be excluded from viable (FITC
negative) and early apoptotic (FITC positive) cells. In the absence of phagocytosis final
stages of apoptosis involve necrotic-like disintegration of the total cell, thus cells in late
apoptosis will be labeled with both FITC and propidium iodide.

MATERIALS REQUIRED:
Equipment : Centrifuge, Vortexer.
Reagent : 10X Annexin V Binding Buffer, 1X binding buffer, FITC Annexin V,
Propidium Iodide Staining Solution (PI), PBS (phosphate buffered saline)
Other Requirements : Micropipettes, eppendorf tubes, Tips, Water bath.
 2

PROCEDURE:
1. Transfer 5x105 cells to a eppendorf tube containing 1mL media.
2. Pellet cells at 1,000 rpm for 4 minutes.
3. Aspirate supernatant and resuspend the cell pellet in 1mL PBS.
4. Pellet cells at 1,000 rpm for 4 minutes.
5. Repeat steps 3 and 4 one more time.
6. Aspirate supernatant, and resuspend cells in 1mL 1X binding buffer to a final concentration
of 2-5 X 105 cells/ ml.
7. Transfer 195 µl cell suspension to a clean tube and add 5 µl Annexin V- FITC.
8. Mix and incubate for 10 min at room temperature.
9. Wash the cells with 1X binding buffer and centrifuge at 1,000 rpm to pellet the cells.
10. Discard the supernatant.
11. Resuspend the cell pellet in 190 µl binding buffer and add 10 µl Propidium Iodide (Final
concentration = 1 µg/ml).
12. Fix the cells in 2% formaldehyde for 10 min. Then place 10 µl of the fixed cell solution in
a slide and place a coverslip over the solution for visualization. (Cells must be incubated with
Annexin V-FITC before fixation since any cell membrane disruption can cause nonspecific
binding of Annexin V to PS on the inner surface of the cell membrane.)
13. Analyze by fluorescence microscopy. Cells that have lost membrane integrity will show
red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell
surface (plasma membrane).

Note:
a. Cells negative for Annexin V and negative for propidium iodide are nonapoptotic.
b. Cells positive for Annexin V and negative for propidium iodide are viable, but undergoing
apoptosis.
c. Cells positive for Annexin V and positive for propidium iodide are dead.

OBSERVATION:

RESULT: