Acridine Orange hydrochloride

This plan only provides a guide, please modify it to meet your specific needs.
1 Preparation of Acridine Orange hydrochloride
(1) Staining solutions: Dissolve Acridine Orange hydrochloride powder in DMSO to 1mg/mL. Aliquot and store unused stock at −20°C or −4°C in the dark (preferably under nitrogen); avoid repeated freeze–thaw cycles.
(2) Working solution: Dilute the stock in an appropriate buffer (serum-free medium, HBSS, or PBS) to 0.5–20μg/mL. Prepare fresh for each experiment and titrate to optimal concentration.
2 DNA/RNA staining of suspended cells
(1) Harvest cells; place ~0.2mL of original suspension into 2–5mL tubes on ice.
(2) Slowly add 0.4mL ice-cold permeabilization buffer; keep on ice for ~15s.
(3) Slowly add 1.2mL ice-cold Acridine Orange hydrochloride working solution; incubate on ice in the dark for 2–15min.
(4) Analyze immediately by flow cytometry: excite at 488nm; collect green emission for dsDNA at 530nm and red emission for RNA/ssDNA at ≥640nm (AO–nucleic-acid complex peaks at 488/650nm).
3 Adherent-cell staining (on ice)
(1) Culture cells on sterile coverslips.
(2) Remove medium, blot excess, and keep coverslips in a humid chamber.
(3) Add 100μL Acridine Orange hydrochloride working solution to one edge; gently tilt to cover all cells. Incubate on ice in the dark for 2–15min.
(4) Aspirate stain, rinse coverslip 2–3 times with fresh medium, and examine under a fluorescence microscope.
4 Detection
(1) Fluorescence microscope or flow cytometer: excite at 488nm; acquire green fluorescence at 530nm (dsDNA) and orange fluorescence at 640nm (RNA/ssDNA).
Precautions:
(1) If the cells need to be stained after fixation, it is recommended to use 70% ethanol for fixation, fix on ice for ≥ 2 hours, rinse with pre-cooled PBS 1-2 times after fixation, remove all ethanol, and then follow the above steps for staining;
(2) There is a quenching problem in fluorescent dyes, please try to avoid light to slow down fluorescence quenching;
(3) For your safety and health, please wear lab coats and disposable gloves when operating.

References:
[1] Hu P, Wang J, Qing Y, et al. FV-429 induces autophagy blockage and lysosome-dependent cell death of T-cell malignancies via lysosomal dysregulation. Cell Death Dis. 2021 Jan 13;12(1):80.