Pyronin Y (Pyronine G) is an intercalating cationic dye with specificity for RNA.

Pyronin Y forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells. The technique has been used to stain RNA in human reticulocytes for subsequent flow analysis and sorting[2]. In viable cells this dye also accumulates in mitochondria. At a concentration of 1.7 to 3.3 μM, pyronin Y is localized almost exclusively in mitochondria of cultured cells, similar to another mitochondria! probe, rhodamine 123. At that concentration PY is not toxic but suppressed cell growth, arresting cells[3].

Instructions:
a. Solution preparation:
1. Mother solution preparation: Prepare a certain concentration of Pyronin Y mother solution, store it at -20℃ or -80℃ away from light after aliquoting.
2. Working solution preparation: Select the appropriate working solution concentration according to the experimental requirements, such as diluting it into a working solution with a concentration of 1 μg/μL, and try to prepare it for immediate use.
b. Operation steps
1. Cell preparation:
Suspended cells: Collect cells by centrifugation, add PBS and wash twice, 5 minutes each time.
Adherent cells: Digest, centrifuge and collect cells, add 1 mL PBS, mix thoroughly and centrifuge again, add 300 μL PBS to resuspend cells. The cell density is controlled at 1×106 cells.
2. Add 1 μL Pyronin Y (1 μg/μL) and place on ice away from light for 20 minutes.
3. Perform flow cytometry analysis on the machine.