Acridine Orange hydrochloride (solution)

Protocol (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock solution preparation
1.1 Solvent: H2O.
1.2 Concentration: Prepare a 1 mg/mL Acridine Orange (AO) stock solution.

2. Working solution preparation
2.1 In complete cell culture medium: Dilute the Acridine Orange stock solution with complete cell culture medium to a final concentration of 2-5 µg/mL. Adjust the working concentration as needed and prepare fresh before use.
2.2 In aqueous phosphate-citric acid buffer: Prepare a buffer containing 37 mM citric acid, 126 mM Na2HPO4, 150 mM NaCl, and 1 mM Na2EDTA. Mix 370 mL 0.1 M citric acid, 630 mL 0.2 M Na2HPO4, 8.77 g NaCl, 340 mg Na2EDTA, 6.0 mL 1 mg/mL Acridine Orange stock solution with H2O to 1 L, so that the AO concentration in the final staining solution is 6 µg/mL. Test 5-20 µg/mL as needed to adjust the working concentration. Store the Acridine Orange staining solution at 4°C protected from light or in a foil-wrapped bottle for up to 6 months.
2.3 For parasite labeling: Use 5 µg/mL, 50 µg/mL, or 100 µg/mL Acridine Orange staining solution. Use a low Acridine Orange concentration to reduce staining of host cell DNA and RNA.

3. Staining procedure
3.1 For adherent primary cells or cell lines^[1]:
3.1.1 Culture cells in DMEM containing 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C, 5% CO2.
3.1.2 Trypsinize the cells and seed them in a black 96-well plate for fluorescence reading at a density that reaches 80-90% confluence on the day of the experiment.
3.1.3 Allow the cells to adhere for at least 24 h.
3.1.4 Remove the medium and add 100 µL of 2-5 µg/mL Acridine Orange staining solution to each well.
3.1.5 Incubate at 37°C for 15 min.
3.1.6 Wash the cells twice with 100 µL complete phenol-free medium for 5 min each.
3.1.7 Place the cells in 100 µL complete phenol-free medium.

3.2 For unfixed cells^[2]:
3.2.1 Use cells suspended in tissue culture medium containing 10% (v/v) serum or 1% (w/v) BSA.
3.2.2 Transfer the cells to a small glass or plastic tube and cool on ice.
3.2.3 Avoid pipetting, vortexing, or mechanically agitating the cells to prevent cell lysis.
3.2.4 Add 0.4 mL ice-cold cell permeabilization solution (0.1% Triton X-100, 80 mM HCl, 150 mM NaCl), keep the cells on ice, and wait 15 sec.
3.2.5 Gently add 1.2 mL ice-cold 6 µg/mL Acridine Orange staining solution and keep the cells on ice protected from light.
3.2.6 Measure and record cell fluorescence within 2-10 min after adding the Acridine Orange staining solution, keeping the samples on ice before and during measurement.

3.3 For fixed cells^[2]:
3.3.1 Rinse the cells once with ice-cold PBS and suspend to approximately 10⁶ cells/mL.
3.3.2 For adherent cells, collect by trypsinization, combine the trypsinized cells with the suspended cells, and rinse once with serum-containing medium, and do not keep on ice for more than 30 min before fixation.
3.3.3 Fix by rapidly injecting 1 mL of cell suspension into 10 mL ice-cold 70% ethanol and fix on ice for ≥2 h.
3.3.4 Before staining, centrifuge the fixed cells at 300 × g for 5 min at 4°C; remove all ethanol; rinse once with ice-cold PBS; transfer the cells to a small tube; and cool on ice.
3.3.5 Avoid pipetting, vortexing, or mechanically agitating the cells to prevent cell lysis.
3.3.6 Add 0.4 mL ice-cold cell permeabilization solution (0.1% Triton X-100, 80 mM HCl, 150 mM NaCl), keep the cells on ice, and wait 15 sec.
3.3.7 Gently add 1.2 mL ice-cold 6 µg/mL Acridine Orange staining solution and keep the cells on ice protected from light.
3.3.8 Measure and record cell fluorescence within 2-10 min after adding the Acridine Orange staining solution, keeping the samples on ice before and during measurement.

3.4 For L. major promastigotes in suspension^[3]:
3.4.1 Label 1 × 10⁶ parasites in suspension.
3.4.2 Add the Acridine Orange staining solution to the parasite suspension.
3.4.3 Incubate the parasite-Acridine Orange mixture for 10 min.
3.4.4 Centrifuge the stained parasites and wash seven times with PBS.
3.4.5 After washing, incubate the stained promastigotes with fibroblasts for 6-8 h.

4. Controls
4.1 Include at least one cell-free well for background fluorescence.
4.2 Include unexposed controls for all conditions used in the experiment.
4.3 Optional positive control 1: 5-50 µg/mL glucose oxidase.
4.4 Optional positive control 2: 0.1-5 mM LLOMe.
4.5 Determine the concentration of LMP inducer applicable to the selected cell type.
4.6 To assess RNA stainability, centrifuge ethanol-fixed cells at 300 × g for 5 min at 4°C, remove all ethanol, resuspend the cells in 1 mL RNase solution (100 µg/mL RNase A in PBS containing Mg²⁺ ), and incubate at 37°C for 30 min. Use DNase-free RNase, or boil the RNase solution for 5 min and cool to 37°C before use.
4.7 To assess DNA stainability, centrifuge the cells as above, resuspend the pellet in 1 mL DNase solution (500 µg/mL DNase I in PBS containing Mg²⁺ ), and incubate at 37°C for 30 min.
4.8 Stain control cells with Acridine Orange as above and assess the percentage loss of red luminescence and green fluorescence as indicators of RNA and DNA staining specificity, respectively.
4.9 For unfixed Acridine Orange-stained cells, after permeabilization and staining, treat with 100 µg/mL RNase A and incubate at 24°C for 30 min, then perform fluorescence measurement.
4.10 Use Acridine Orange-stained free fibroblasts on day 3 of culture as a control for comparison with infected fibroblasts.

5. Detection and analysis
5.1 Instrument types:
5.1.1 Fluorescence plate reader, flow cytometer, fluorescence microscope.

5.2 Wavelength settings:
5.2.1 Plate reader, green fluorescence: excitation 485 nm, emission 535 nm. Red fluorescence: excitation 465 nm, emission 650-710 nm.
5.2.2 Flow cytometry, DNA detection: excite at 488 nm and collect green fluorescence at 530 nm. The AO-dsDNA absorption peak is 502 nm; recommended excitation 488 nm, emission 520-524 nm, using a 520 nm filter.
5.2.3 Flow cytometry, RNA detection: preferably collect red luminescence above 640 or 650 nm. For dual-laser excitation, use 488 nm for DNA green fluorescence detection and 457 nm for RNA red luminescence detection. AO-ssDNA and AO-ssRNA absorption is 426-458 nm; recommended excitation 457 nm, emission 630-644 nm, using a >640 nm filter.
5.2.4 Fluorescence microscopy: green fluorescence maximum emission wavelength is 525 nm; AO bound to dsDNA emits green fluorescence at 525 nm, and AO bound to RNA or ssDNA emits red fluorescence at 650 nm.

5.3 Measurement and analysis:
5.3.1 Perform a single-point measurement to record the initial AO fluorescence before induction of LMP.
5.3.2 Begin acquiring fluorescence data immediately after adding the LMP inducer at 37°C, using the same settings as the initial measurement.
5.3.3 For LLOMe, acquire fluorescence intensity data once per minute, as maximum leakage typically occurs within 30 min.
5.3.4 For glucose oxidase, acquire data every 2-3 min for up to 2 h.
5.3.5 Control cells show a green monomeric AO emission peak at 535 nm and a red dimeric lysosomal emission peak at 650 nm.
5.3.6 Use the increase in AO green fluorescence as a more sensitive readout of changes in the lysosomal proton gradient, because the decrease in red intensity is less easily detected.
5.3.7 AO intercalated into dsDNA emits green fluorescence, whereas AO bound to ssRNA emits red luminescence with a maximum emission wavelength above 630 nm.
5.3.8 DNA frequency histograms from green fluorescence can be deconvoluted to obtain G1 vs S vs G2/M ratios.
5.3.9 Live promastigotes appear green. After 12 h of culture, free promastigotes appear green before entering fibroblasts.
5.3.10 AO-stained fibroblast nuclei are partially green (approximately 12 μm), nucleoli are green (approximately 0.6-1 μm), and parasites (2.5-5 μm) can be distinguished from nucleoli by size.