Linkage Biological Apoptosis Kit AP101 Detection Procedure

Annexin V/PI apoptosis kit (Cat#: AP101) is a kind of apoptosis of phosphatidylserine which appears on the surface of cell membrane when FITC-labeled recombinant human Annexin V is used to detect apoptosis. Detection kit. It can be detected using flow cytometry, fluorescence microscopy or other fluorescence detection equipment.

The kit also provides propidium iodide (PI) staining solution. PI can stain necrotic cells or cells that lose cell membrane integrity in the late stage of apoptosis, showing red fluorescence. For necrotic cells, since the integrity of the cell membrane has been lost, Annexin V-FITC can enter the cytoplasm and bind to phosphatidylserine located inside the cell membrane, thereby also causing the necrotic cells to exhibit green fluorescence. Therefore, by matching Annexin V with PI, cells at different times can be distinguished.

Kit components

Catalog number / specification

Component

AP101-30

AP101-60

AP101-100

30T

60T

100T

Annexin V-FITC

150ul

300ul

500ul

5x Binding Buffer

5ml

10ml

15ml

Propidium Iodide (PI)

300ul

600ul

1000ul

Kit step

Sample processing

    (1) Inducing apoptosis by a suitable method;

    (2), suspension cells were centrifuged (1500 rpm for 5 min) to collect; adherent cells were digested with trypsin without EDTA and washed once with serum-containing medium (Note: trypsin digestion time is not too long, otherwise it is easy to cause false positive); Or directly with Accutase enzyme digestion (without termination); centrifugation at 1500 rpm for 5 min to collect cells;

    (3), washing the cells twice with PBS (centrifugation at 1500 rpm for 5 min) to collect 1-5 X105 cells, discard the supernatant;

    (4) Prepare 1X Binding Buffer buffer: Calculate the amount of 10 times, add 1ml of 5X Binding Buffer buffer to 4ml of deionized water;

    (5) Resuspend the cells with 500ul 1X Binding Buffer (buffer diluted in step 4)

    (6), add 5ul annexin-V FITC and 10ul PI

    (7) Incubate for 5 minutes at room temperature

    (8), machine detection

Flow detection

Flow analysis Annexin-V FITC (radiation wavelength = 488 nm; absorption wavelength = 530 nm) is detected by FITC signal (usually FL1 channel); PI dye solution is detected by PE signal (usually FL2 channel)

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