Question

Can you write about the function of the following reagents in the flow cytometry – Annexin V/PI staining:

1. PBS

2. 1X Binding buffer: 10 mM Hepes pH 7.4, 140 mM NaCl and 2.5 mM CaCl2 (10X dilution before use)

3. Annexin V-FITC staining solution

4. Propidium iodine (PI) staining solution

Thank you in advance, I would appreciate it a lot!!!

Answer 1

1. PBS (phosphate-buffered saline) is a buffer solution which is generally used to wash cells while preparing samples for annexin V/PI staining. PBS is always used when working with cell culture as it maintains a constant pH and thus is strongly recommended when washing cells to remove unwanted cell culture media.  

2. Binding buffer is an isotonic solution which is necessary for the binding of Annexin V to phosphatidylserine.  Annexin V binding is very sensitive to the buffer components.

3. The phenomenon of apoptosis (a type of programmed cell death) involves flipping of the plasma membrane components. During apoptosis, the phosphatidylserine (a phospholipid) which is preferentially present on the inner side (towards cytosol) of the plasma membrane bilayer is translocated towards the outer side. Annexin V-FITC specifically binds with this phospholipid and thus helps in the identification of those cells which died due to apoptosis.

4. Necrotic cells are specifically stained with Propidium Iodide and thus it helps in the identification of cells that died due to necrosis.

The above figure shows flow cytometric determination of the apoptotic potential of a drug by Annexin V-FITC/PI assay in cancer cells. From four sub-quadrants of each scattergram, lower left indicates live cells neither stained by Annexin V or PI, upper left indicates necrotic cells specifically positive for PI, while lower right is particular for Annexin V (indicates cells in the early apoptosis) and upper right quadrant for both the Annexin V and PI indicates late apoptotic stages.