Operational Guidelines for Annexin V Fluorescent Double-staining Apoptosis Detection Kit


Elabscience® Annexin V Fluorescent Double-Staining Cell Apoptosis Detection kit was used to identify apoptotic and necrotic cells.

Annexin V is a member of the annexin family of intracellular proteins that bind to phosphatidylserine (PS) in a calcium-dependent manner.  Annexin V-Flouorescent, which bind to the membrane of apoptotic cells through PS exposed outside cells, can detect apoptosis by flow cytometry or fluorescence microscopy.

Specifical Staining Buffer which can specifically bind to double-stranded DNA and produces strong fluorescence, and it's normally fails to penetrate cell membranes. Due to late apoptotic or necrotic cells loss of integrity of membrane, Specifical Staining Buffer can enter the cells to stain DNA. Cells at different apoptotic stages can be distinguished by using with Annexin V and Specifical Staining Buffer.

1. Suspension Cells

A. Suspension cells were induced to apoptosis according to the experimental scheme. Centrifuge at 300 g for 5 min, discard the supernatant. Add appropriate Cell Staining Buffer [Cat: E-CK-A107, please contact the local distributors] to re-suspend gently and count the cells.

B. Take 1-5 × 105 re-suspend cells in step1. Centrifuge at 300 g for 5 min, discard the supernatant. Add appropriate Cell Staining Buffer [Cat: E-CK-A107] to wash the cells, centrifuge at 300 g for 5 min, discard the supernatant. Add 500 μL of 1 × Annexin V Binding Buffer to re-suspend the cells.

C. Add 5 μL of Annexin V-Fluorescent Staining Buffer and 5 µl of DNA dye to each tube.

D. Gently vortex the cells and incubate at room temperature for 15-20 min in the dark.

E. Analyze the cells immediately with proper machine settings. Otherwise, place the cells on ice in the dark and analyze within 1 h.

F. Detection

1) Flow cytometry detection

The processed sample is tested on the flow cytometer.

2) Fluorescence microscope analysis

Take a drop of cell suspension double-stained with fluorescein-Annexin V/nucleus staining solution (the cell suspension treated in step E) on a glass slide, and cover the cells with a cover glass. Observe with a two-color filter under a fluorescence microscope.

2. Adherent cells

1) Flow cytometry detection

A. Perform apoptosis induction according to the experimental protocol. Aspirate and t ransfer the medium to a clean centrifuge tube (standby), and wash the culture flask cells once with PBS free of calcium and magnesium ions.

B. Add an appropriate amount of trypsin digested cells to the culture flask, and incubate at room temperature until gently pipetting to detach the adherent cells. Aspirate the trypsin digestion solution to avoid excessive trypsin digestion.

Note: For adherent cells, the trypsinization step is critical. If the trypsin digestion time is too short, the cells need to be blown off to fall off, which will easily cause damage to the cell membrane, leading to false positives of cell necrosis; if the digestion time is too long, it is also likely to cause cell membrane damage and false positives of cell necrosis, or even Affect the binding of phosphatidylserine and Annexin V-fluorescein on the cell membrane to interfere with the detection of apoptosis. At the same time, try not to contain EDTA in the trypsin cell digestion solution, because EDTA may affect the binding of Annexin V and phosphatidylserine.

C. Add the cell culture medium collected in step A, gently blow down the cells, transfer to a centrifuge tube, centrifuge at 300g for 5 minutes, discard the supernatant, collect the cells, wash the cells once with PBS, gently suspend the cells and count them.

Note: It is very important to add cell culture fluid. On the one hand, it can collect suspended cells t hat have undergone apoptosis or necrosis. On the other hand, the serum in the cell culture fluid can effectively inhibit or neutralize residual pancreatin (the residual pancreatin will digest And degrade the annexin V-fluorescein added subsequently, causing staining failure).

D. Take 1~5×105 resuspended cells, centrifuge at 300g for 5min, discard the supernatant. Add 500μL of Annexin V Binding Buffer diluted to 1 × to resuspend the cells.

E. Add 5μL of fluorescein labeled Annexin V and 5μL of nuclear staining solution to the cell suspension.

F. After gently vortexing to mix, incubate for 15-20 minutes at room temperature in the dark.

G. Test on the machine immediately after the reaction is completed. If the test cannot be performed in time, please place it on ice and avoid light and complete the test within 1 hour.

H. Flow cytometry detection

2) In-situ detection by fluorescence microscope

Note: The advantage of this method is that cell apoptosis can be observed in situ, but the disadvantage is that some apoptosis cannot be detected due to weak adhesion.

A. If possible, after inducing apoptosis, centrifuge at 300g for 5 minutes in a multi-well plate centrifuge.

B. Aspirate the cell culture medium and wash twice with PBS (if possible, do step A before).

C. Discard the supernatant after centrifugation, and add 500 μL of Annexin V Binding Buffer working solution diluted to 1 ×.

D. Add 5μL of fluorescein-labeled-Annexin V and 5μL of nuclear staining solution.

E. Mix gently with a pipette, and incubate for 15-20 minutes at room temperature in the dark.