PI, or propidium iodide, is an analog of ethidium bromide, which can bind strongly to DNA. It releases red fluorescence after being inserted into double-stranded DNA to achieve staining of DNA or cell nuclei. PI cannot penetrate the intact cell membrane, but can penetrate the damaged cell membrane of late apoptotic cells and dead cells. Taking advantage of this feature, PI is usually used in combination with fluorescent probes such as Calcein-AM or FDA to stain and observe live and dead cells. Or use flow cytometry to detect cell apoptosis and cell cycle quantitatively. The maximum excitation wavelength of the PI-double-stranded DNA complex is 535nm, and the maximum emission wavelength is 615nm.
The PI staining solution of this product is ready-to-use, with a PI concentration of 100 μg/mL, which can be directly used for the nuclear staining of necrotic cells or tissues, and the cell suspension can be stained with this staining solution for cell cycle detection by flow cytometry .
Operation steps for flow cytometry to detect cell cycle:
1. The cultured live cells are digested, washed with PBS, and collected by low speed centrifugation to obtain cell pellets.
2. Slowly add 1-3 mL of 90% ethanol pre-cooled at -20°C to the cell pellet, resuspend the cells in an ice bath overnight.
3. Centrifuge at 1500 rpm for 5 minutes to collect the cells, resuspend in PBS, and centrifuge again to remove the supernatant.
4. Add 250 μL PBS to resuspend the cells.
5. Add 2 μL of RNase A (recommended WGR8021) at a concentration of 1 mg/mL, and water bath at 37°C for 40 min.
6. Add 50 µL of PI staining solution and incubate in the dark for 20 minutes (the time can be adjusted according to the staining results of the experimental materials).
7. Flow cytometry detection.
Operation steps of fluorescence microscope observation of living and dead cells:
1. After the cultured adherent cells, remove the medium and wash twice with PBS.
2. Dilute the PI staining solution 10-20 times with PBS to prepare a PI staining solution with a final concentration of 5-10 μg/mL. Add an appropriate amount of PI staining working solution to the well to cover the cells, and incubate for 5-10 min at room temperature in the dark.
3. Discard the staining work, add PBS to cover the cells, and observe with a fluorescence microscope. The nuclei of dead cells or late apoptotic cells show red fluorescence.
1. Fluorescent dyes have the problem of quenching. It is recommended to complete the test on the same day after dyeing.
2. Please wear lab coat and disposable gloves during operation.
Packing and Shipping
All of our products will be packed in standard carton.