1. Introduction

Hello Bio's Annexin V-PE Apoptosis Staining / Detection Kit identifies early apoptotic, necrotic and viable cells. Annexin V-PE is a calcium dependent fluorescent probe used to identify apoptotic cells. Annexin V has a high affinity for the phosphatidylserine molecules that translocate from the inner to outer facing side of the cell membrane in cells that are undergoing apoptosis. 7-Aminoactinomycin D (7-AAD) is a DNA binding fluorescent probe that is cell membrane impermeant in healthy cells. By looking at the staining pattern of Annexin V-PE and 7-AAD it is possible to identify the cell fate:

• Cells showing Annexin V-PE staining only are currently undergoing apoptosis.
• Cells showing both Annexin V-PE and 7-AAD staining are either end stage apoptotic, necrotic or already dead.
• Cells showing neither Annexin V-PE nor 7-AAD staining are currently alive and not undergoing apoptosis.

This kit is designed for use in measuring apoptosis in non-adherent cells for flow cytometry and fluorescence microscopy alongside adherent cells for fluorescence microscopy. Please note that the use of this kit for measuring apoptosis using flow cytometry in adherent cells is not recommended due to membrane damage during cell detachment.

 

This kit contains:

• Annexin V-PE - 0.5ml
• 7-AAD solution - 2ml
• Annexin V binding buffer (10x) - 5ml

Note: Please ensure that all components are only used in sterile conditions to prevent contamination of the stock solutions.

 

This kit additionally requires:

• Microcentrifuge
• Sterile PBS
• 5ml sterile cell culture tubes
• Vortex mixer and flow cytometer (if analysing by flow cytometry)
• Rocker, 22mm coverslips, microscope slides, paraformaldehyde and fluorescence microscope (if analysing cells by fluorescence microscopy)

2. Protocol

2.1 Annexin V-PE Assay - Flow cytometry

Annexin V-PE is commonly used to measure apoptosis in non-adherent cells using flow cytometry. In order to set up compensation and quadrants it is recommended to set up the following control conditions:

• Unstained cells
• Cells stained with only 7-AAD
• Cells stained with only Annexin V-PE

When inducing apoptosis it is recommended to normalise results to an untreated population due to there always being a basal level of apoptosis and necosis in any cell population. It is also recommended to also use a positive control such as 2µM Camptothecin.

Protocol for flow cytometry:

1. Culture cells using normal protocols.
2. Pellet cells through centrifugation then wash twice with ice-cold sterile PBS.
3. Pellet cells again then using a 5ml sterile cell culture tube resuspend the cells in 100µl of 1x binding buffer such that 100µl contains 1x10⁵ cells
4. Add 5µl of Annexin V-PE and 5µl of 7-AAD to the cells, gently vortex then incubate at room temperature for 15 minutes in the dark.
5. Add 400µl of 1x binding buffer and then analyse by flow cytometry within an hour.
6. Analyze Annexin V-PE (Excitation: 488nm, Emission: 578nm) and 7-AAD (Excitation: 488nm, Emission: 650nm (FL3 channel)) binding using suitable filtersets installed on the flow cytometer.

2.2 Annexin V-PE Assay - Fluorescence Microscopy

Annexin V-PE can also be used to measure apoptosis in both adherent and non-adherent cells using fluorescence microscopy. When using Annexin V-PE in fluorescence microscopy it is always recommended to make use of both negative controls (untreated cells) and positive controls (e.g. 2µM Camptothecin)

Protocol for adherent cells:

1. Culture cells on 22mm coverslips to a density of 1x10⁵ cells per coverslip
2. Wash cells twice with ice-cold sterile PBS.
3. Incubate cells with 500µl of 1x binding buffer
4. Add 5µl of Annexin V-PE and 5µl of 7-AAD to the coverslip then gently rock for 15 minutes at room temperature in the dark.
5. Either image cells directly or wash in binding buffer then fix with 2% formaldehyde
6. Image Annexin V-PE with a RHOD filter set and 7-AAD (Ex: 488nm, Em: 650nm) using appropriate filters.

Protocol for non-adherent cells:

1. Culture cells using normal protocols.
2. Pellet cells through centrifugation then wash twice with ice-cold sterile PBS.
3. Pellet cells again then using a 5ml sterile cell culture tube resuspend the cells in 100µl of 1x binding buffer such that 100µl contains 1x10⁵ cells
4. Add 5µl of Annexin V-PE and 5µl of 7-AAD to the cells, gently vortex then incubate at room temperature for 15 minutes in the dark.
5. Add the cells to a glass microscope slide then coverslip and image Annexin V-PE with a RHOD filter set and 7-AAD (Ex: 488nm, Em: 650nm) using appropriate filters..
   1. Optional: Before adding to the microscope slide wash with 1x binding buffer then fix with 2% formaldehyde.

2.3 Interpretation

• Cells showing Annexin V-PE staining only are currently undergoing apoptosis.
• Cells showing both Annexin V-PE and 7-AAD staining are either end stage apoptotic, necrotic or already dead.
• Cells showing neither Annexin V-PE nor 7-AAD staining are currently alive and not undergoing apoptosis.

2.4 Troubleshooting

Please follow the below table to resolve any problems encountered when using this kit. For any problems not listed or for any further advice please contact our technical support team at technicalhelp@hellobio.com

Problem	Potential Cause
High background	Cell density is too high. Try reducing the concentration of cells and repeat the experiment.
	Too long incubation of cells with Annexin V-PE. Try reducing the incubation time and repeat the experiment.
	Use of overly confluent cells. Try repeating the experiment with cells at 80-95% confluency.
	Cells are contaminated (e.g. mycoplasma infection). Test cells for contamination or repeat again with a fresh aliquot of cells.
Low signal levels	Too few cells used in experiment, repeat again using a higher number of cells
	Cells did not initiate apoptosis. If a positive control was not carried out then make sure that this is included in the next experiment. Check the time course of expected apoptosis and ensure that the Annexin V-PE is added at a time point where significant apoptosis would be expected.
	Cells were washed with PBS instead of binding buffer. Ensure that cells are only washed in binding buffer before and after addition of the Annexin V-PE. This includes before and after fixation for adherent cells.
Erratic staining	Ensure that Annexin-V PE staining is carried out before fixing cells. Fixation can make cell membranes leak therefore make results unreliable.
	Adherent cells used for flow cytometry experiments can be unreliable due to membrane damage during detachment. However authors such as van Engeland et al., 1996 and Casciola-Rosen et al., 1996 have reported successful use of adherent cells in flow cytometry when stained for Annexin V-PE.