Annexin V detection of apoptotic cell membrane changes in the operational procedures

I. Annexin V detects changes in apoptotic cell membranes

Reagents and solutions:

A For separately purchased reagents:

a) 10X Binding Buffer (Cat. No. 556454)
b) Propidium Iodide (PI, Cat. No. 556463): It is recommended to use Annexin V-FITC (Cat. No. 556420, 556419) or Annexin V-Biotin (Cat. No. 556418, 556417) with the following reagents.
c) Via-Probe? (Cat. No. 555816): Nucleic acid dye 7-AAD. It is recommended to use Annexin V-PE (Cat. No. 556422,556421) or Annexin V-Biotin (Cat. No. 556418, 556417) with the following reagents.
d) 1X PBS Buffer (Cat. No. 554781): 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO 4 7H20, 0.24 g KH2PO4, add water to 1 L. Adjust the pH to 7.2. Autoclave was stored at room temperature.

B. For the apoptosis detection kit (Cat. No. 556547)

a) FITC Annexin V (component number: 51-65874X): 5 μl per sample.
b) Propidium Iodide (PI, component number: 51-66211E)): Ready-made nucleic acid dyes. A dose of 5 μl per sample.
c) 10X Annexin V Binding Buffer (component number: 51-66121E)

Steps:

1. Take the Falcon tube and edit the negative control tube and specimen tube number in the order of the specimen.
2. Wash the cells twice with cold PBS buffer and make a 1x106 cells/ml suspension with 1x Binding Buffer buffer.
3. Add 100 μl of cell suspension to the Falcon tube.
4. Add AnnexinV and Nucleic Acid Dyes in the following volumes:

5. Mix gently and leave at room temperature (20 ° C ~ 25 ° C) for 15 minutes.
6. * When testing with Annexin V-Biotin reagent:
● Wash the cells once with 1xBinding Buffer buffer and remove the supernatant.
● 100 μl of 1x Binding Buffer buffer was dissolved in 0.5 μg of SAv-FITC reagent and added to the cell tube.
● Mix gently. Add 5 μl of PI and leave it at room temperature (20-25 ° C) for 15 minutes in the dark.
7. 400 μl of 1×Binding Buffer buffer was added to each test tube. The results were measured by an up-flow cytometer within 1 hour.

Annexin V Blocking

Pre-incubation of the cells to be tested with unlabeled recombinant Annexin V followed by experiments was the quality control of apoptosis detection of Annexin V cells. The principle is to block the binding site of Annexin V-FITC in advance, which can prove the specificity of Annexin V-FITC apoptosis assay.

1. Wash the cells twice with cold PBS buffer and make a 1x106 cells/ml suspension with 1x Binding Buffer buffer.
2. Add 100 μl of cell suspension (approximately 1 x 105 cells) to a 5 ml culture tube.
3. Add 5-15 μg of purified recombinant Annexin V. Note that different cell lines, as well as different stages of apoptosis, require different levels of purified recombinant Annexin V for their Annexin V site saturation. In some cases, to achieve the best results, the number of cells can be reduced by adding 5-15 μg of purified recombinant Annexin V to 0.5 x 105 cells.
4. Mix gently and allow to react at room temperature for 15 minutes.
5. Add 5 μl of Annexin V-FITC or / and PI, mix gently, and leave at room temperature for 15 minutes in the dark.
6. Add 400 μl of 1x Binding Buffer buffer to each test tube.
7. Up-flow cytometry results within 1 hour.

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