This experimental method was obtained from the official website of the Fourth Military Medical University

Operation method

Sample preparation experiments for DNA fluorescence staining

Materials and Instruments

PBS
DNA fluorescent dye Flow cytometry

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DNA is a relatively constant parameter of intracellular content that changes with each time phase of the cell proliferation cycle. Fluorescent dyes (e.g., PI) selectively and quantitatively embed themselves between the bases of the double helix of nucleic acids (DNA/RNA) and bind specifically to the cell, and the DNA content is directly proportional to the amount of the fluorescent dye bound, so that cell proliferation can be obtained by measuring fluorescence intensity.

1. Centrifuge the fixed cells (500~1000rpm, 5min) and discard the supernatant, then wash with PBS twice.

2. Adjust the cell concentration with PBS to 1×106 cells/100μl per portion.

3. Add 1000 μl of DNA fluorescent dye (usually with the DNA staining kit provided by Coulter), and stain for 15 min at room temperature, protected from light.

4. Detect on flow cytometer.

Sample preparation can analyze the percentage of each time phase of the cell cycle, and at the same time can roughly observe the phenomenon of apoptotic cells, if the control solution (chicken erythrocytes) is used as a reference standard, it can be used for cellular DNA ploidy analysis, and the relative content of DNA can be measured by the DNA index (DNA index), and the DI can be calculated by the following formula:

DI = average value of G0/G1 phase of the sample / average value of G0/G1 phase of normal diploid cells

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