Cell Function
Annexin V Fluorescence Double Staining Apoptosis Detection Kits Experimental Operation Guide
Source: Elabscience®Published: Jul 22,2024
The fluorescence double staining apoptosis detection kits developed by Elabscience® are used to identify apoptotic and necrotic cells.
Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). It binds to PS exposed on the outer membrane of early apoptotic cells. Annexin V labeled with a fluorescent dye can be used with flow cytometry or fluorescence microscopy to detect apoptotic cells.
Due to the loss of membrane integrity in late-stage apoptosis or necrotic cells, nuclear staining solution can enter the cells and stain the nuclei. This can differentiate cells at different stages of apoptosis when used in conjunction with Annexin V.
A. Suspension Cells:
1. Follow the experimental protocol for apoptosis induction, centrifuge at 300 g for 5 min, discard the supernatant, collect cells, wash cells once with PBS, gently resuspend cells, and count.
2. Take 1~5 × 105 resuspended cells, centrifuge at 300 g for 5 min, discard the supernatant. Add 500 μL 1×Annexin V Binding Buffer to resuspend cells.
3. Add 5 μL of Annexin V and 5 μL of nuclear staining solution to the cell suspension.
4. Gently mix, then incubate at room temperature in the dark for 15~20 min.
5. Immediately proceed to machine detection after completion of reaction. If immediate detection is not possible, incubate on ice in the dark and complete detection within 1 hour.
6. Detection:
1) Flow cytometry analysis
Process the prepared samples on a flow cytometer.
2) Fluorescence microscopy analysis
Take a drop of cell suspension stained with fluorescent-labeled Annexin V/nuclear staining solution (prepared according to steps 4) on a slide, cover with a coverslip, and observe under fluorescence microscopy.
B. Adherent Cells:
I. Flow cytometry detection
1. Follow the experimental protocol for apoptosis induction. Transfer the culture medium to a clean centrifuge tube (set aside), wash the adherent cells in the culture bottle once with calcium- and magnesium-free PBS.
2. Add an appropriate amount of trypsin to digest cells in the culture bottle, and incubate at room temperature until gentle tapping loosens adherent cells. Remove the trypsin digestion solution, avoiding excessive digestion.
Note: Trypsin digestion is crucial for adherent cells. Too short a digestion time may require vigorous tapping to detach cells, potentially damaging the cell membrane and leading to false-positive necrosis. Too long a digestion time can also damage the cell membrane, affect the binding of phosphatidylserine with Annexin V-fluorescein, and interfere with apoptosis detection. Trypsin cell digestion solution should ideally not contain EDTA, as EDTA may affect the binding of Annexin V to phosphatidylserine.
3. Add the collected cell culture medium from step 1, gently tap down adherent cells, transfer to a centrifuge tube, centrifuge at 300 g for 5 min, discard the supernatant, collect cells, wash cells once with PBS, gently resuspend cells, and count.
Note: Adding cell culture medium is crucial to collect floating apoptotic or necrotic cells and serum in the cell culture medium can effectively inhibit or neutralize residual trypsin (residual trypsin can digest and degrade subsequently added Annexin V-fluorescein, leading to staining failure).
4. Take 1-5 × 10^5 resuspended cells, centrifuge at 300 g for 5 min, discard the supernatant. Add 500 μL of Annexin V Binding Buffer diluted to 1× to resuspend cells.
5. Add 5 μL of fluorescent-labeled Annexin V and 5 μL of nuclear staining solution to the cell suspension.
6. Gently vortex and mix, then incubate at room temperature in the dark for 15-20 min.
7. Immediately proceed to machine detection after completion of reaction. If immediate detection is not possible, incubate on ice in the dark and complete detection within 1 hour.
8. Flow cytometry detection.
II. Fluorescence microscopy in situ detection
Note: This method allows for in situ observation of cell apoptosis, but may not detect some apoptotic cells due to weak adherence.
1. If conditions allow, centrifuge with a multi-well plate centrifuge at 300 g for 5 min after inducing apoptosis.
2. Remove the cell culture medium, wash twice with PBS (if possible, perform step 1 before this).
3. Discard the supernatant after centrifugation, add 500 μL of Annexin V Binding Buffer diluted to 1×.
4. Add 5 μL of fluorescent-labeled Annexin V and 5 μL of nuclear staining solution.
5. Gently mix with a pipette, incubate at room temperature in the dark for 15-20 min.
6. Wash cells once with Annexin V Binding Buffer working solution.
7. Fluorescence microscopy detection. Observe under fluorescence microscopy using dual-color filters. (If observation is not possible, place a coverslip over the well plate to allow cell adherence, and then observe after immersion with Annexin V Binding Buffer.)
8. Immediately observe after completion of reaction to avoid cell drying.