Annexin V-FineTest®647/7-AAD Apoptosis Kit

K077

20T/50T/100T

2-8°C for 12 months. Annexin V-FineTest®647 and 7-AAD need to be stored away from light.

ice pack

Annexin V-FineTest®647/7-AAD apoptosis detection kit can be used to detect apoptosis in suspension cells and adherent cells.

Annexin V is a calcium-dependent phosphatidylserine binding protein with a high affinity for phosphatidylserine PS. Annexin V labelled FineTest®647 can bind to the membrane of early apoptotic cells by means of the PS exposed outside the cells. Apoptosis can be detected by flow cytometry or fluorescence microscopy.

7-AAD binds specifically to double-stranded DNA and produces strong fluorescence, which is normally unable to penetrate cell membranes. Due to late apoptotic or necrotic cell membrane loss of integrity, 7-AAD can enter cells to stain DNA and, when used in combination with Annexin V, distinguish cells at different stages of apoptosis.

The following figure shows the apoptosis effect of Jurkat cells induced by camptothecin detected by this kit.

Jurkat cells were treated with 5μM Camptothecin (left) or untreated (right) for 4 h. After staining with this kit, fluorescence detection was performed by flow cytometry. Annexin V-FineTest®647 monopositive cells are early apoptotic cells, Annexin V-FineTest®647 and 7-AAD double positive cells are necrotic or late apoptotic cells, and 7-AAD monopositive cells are nude cells.

A. Incubation of cells with Annexin V-FineTest®647

1. Induce apoptosis by desired method. Centrifuge at 300 g for 5 min, discard the supernatant, collect the cells, gently suspend the cells with PBS and count them.

2. Collect 1-5 x 105 cells by centrifugation, and the supernatant was discarded. The the cells were washed with PBS once, the supernatant was abandoned after centrifugation.

3. Resuspend cells in 500 µl of 1X Binding Buffer.

4. Add 5 µl of Annexin V-FineTest®647 and 5 µl of 7-AAD.

5. Incubate at room temperature for 15 min in the dark.

Proceed to B or C below depending on method of analysis.

B. Quantification by Flow Cytometry

Analyze Annexin V-FineTest®647 binding by flow cytometry using APC signal detector and 7-AAD staining by the PerCP/Cy5.5 detector.

For adherent cells, gently trypsinize and wash cells once with serum-containing media before incubation with Annexin V-FineTest®647 (A.3-5).

C. Detection by Fluorescence Microscopy

Place the cell suspension from Step A.5 on a glass slide. Cover the cells with a glass coverslip.

For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on glass slide and visualize cells.

1.It should be detected as soon as possible after staining. Too long a time may lead to an increase in the number of apoptotic or necrotic cells.

2.When detecting adherent cells, the suspension cells generated after inducing apoptosis should be collected and detected together with the adherent cells collected later.

3.Mechanical damage caused by digestive adherent cells should be avoided as much as possible. At the same time, the digestive juices of pancreatic enzymes should be as free of EDTA as possible, as EDTA affects Annexin V binding to phosphatidylserine.

4.If EDTA-containing pancreatic enzymes are used, the cells should be thoroughly washed after collection to ensure that EDTA is removed.

5.Fluorescent substances are prone to quenching, in the fluorescence observation, as far as possible to shorten the observation time, while in the operation and storage process also try to pay attention to the preservation of light.

6.For your safety and health, please wear a lab coat and disposable gloves.

7.This kit is for scientific research only.