Dmra2

The DC2.4 cells were seeded in 24‐well plates (5 × 10⁴ cells/well) and incubated for 24 h. Next, the cells were stained for 1 h with 5 μg/ml acridine orange dye solution. After removing the dye, the cells were cultured for 24 h with PBS, OVA, Fe₃O₄@Ca/MnCO₃ + OVA, Fe₃O₄@Ca/MnCO₃/OVA, Fe₃O₄@Ca/MnCO₃/OVA(M), or Alum/OVA formulations. The distribution of acridine orange in the cells was observed with a fluorescence microscope (DMRA2, Leica, Germany).
The DC2.4 cells were seeded in 96‐well black plates (1 × 10⁴ cells/well) and incubated for 24 h at 37°C. The treatment of acridine orange staining was as same as the above steps. Then, the red (620 nm) and green (485 nm) fluorescence intensities of the cells were detected with a multifunctional microplate reader (Cytation5, Biotek, USA). Lysosome integrity of the treated cells (%) = (red/green fluorescence intensity of the treated cells)/(red/green fluorescence intensity of the control cells) × 100%.

NIH3T3 cells were cotransfected with pcDNA-HA-PRAK, pcDNA3-Flag-DJ-1, and PEGFP-DJ-1 for 24 hrs. PRAK^+/+ cells and PRAK^−/− cells were starved for 48 hrs and were further challenged with H₂O₂ (300 μM) for 6 hrs. Cells were fixed in 4% paraformaldehyde for 10 min, washed twice with PBS, and permeabilized with 0.1% sodium tetrahydroborate for 5 min. After being washed three times with PBST (PBS + 0.2%Triton X-100) and blocked with 3% BSA for 1 hr, cells were incubated with the indicated antibodies diluted in 3% BSA at room temperature for 1 hr and washed three times with PBST. Cells were further incubated with the indicated secondary antibodies diluted in 3% BSA at room temperature for 1 hr and washed three times with PBST. Cell nucleus was stained with 10 μM DAPI. Fluorescent images were recorded and analyzed using a fluorescence microscope (DMRA2, Leica) equipped with FW4000 software.

Cultures were grown in Yeast Peptone Dextrose Adenine (YPDA: 2% (w/v) peptone, 1% (w/v) yeast extract, 2% (w/v) glucose, 100 µg/ml Adenine; to make snu114∆/snu114∆ diploid shuffle strain) or drop out media (to select for plasmids) overnight to stationary phase. 20 ml YPA (pre-sporulation medium, 2% (w/v) peptone, 1% (w/v) yeast extract, 1% (w/v) potassium acetate) cultures were inoculated in 100 ml flask to OD₆₀₀ 0.10–0.15 and grown with good aeration for 13–14 hours. Cultures with OD₆₀₀ between 1.6–3.0 were harvested and washed with H₂O, re-suspended in 10 ml SPM (Sporulation medium, 1% (w/v) potassium acetate, 0.002% (w/v) raffinose) to an OD₆₀₀ of 1.8–1.9 and incubated at 30ºC shaking vigorously (250 rpm). After 4 hours in SPM, 100 µl of cells were removed every hour and fixed with 400 µl of 95% ethanol. Cell pellets were then stored at 4°C. Tetrads were stained with DAPI and monitored using microscope (Leica DMRA2). For RNA extraction, volumes were scaled up. For tetrad dissection 75 µl cell culture in sporulation medium with an OD₆₀₀ of 2.0, was pelleted and resuspended in 18 µl of 1 M sorbitol, and Zymolyase was added to 1 mg/ml Following incubation for 10 min at 20°C, 400 µl of ice cold water was added and stored on ice until required. Tetrads were dissected using Micromanipulator (Singer Instruments MSM System).

Mid-late 3^rd instar larvae were dissected in either 1× PBS or Grace's solution, and fixed in 6% formaldehyde as previously described [98] (link), and immunolabeled using anti-cleaved-Caspase-3, 1∶50 (Cell Signaling). Secondary antibody was anti-rabbit 488 at 1∶500 dilutions, and DNA was counterstained with DAPI. X-gal staining was performed 6 hours after heat shock treatment as previously described [19] (link). TUNEL staining (In Situ cell death detection kit, TMR red, Roche) was performed according to manufacture's instructions. Wide-field micrographs were taken on a Leica DMRA2 and analyzed using OpenLab (Improvision) software. Confocal micrographs were captured on a Leica SP5 confocal.

To estimate total cells and neuron numbers from the dissected brain parts, we followed the isotropic fractioned method^{66 (link)}. The method consisted of dissociating brain tissue using a tissue grinder. To facilitate dissolving brain cell membranes while simultaneously ensuring that nuclear membranes remain intact, we ground the tissue in a saline detergent solution. Upon tissue homogenisation, samples were stained with diamino-phenyl-indol (DAPI). In a first step, cell counts were performed for every brain part with a haemocytometer (Brand® counting chamber Blaubrand® Neubauer improved) under a microscope with fluorescence (Leica DMRA2) using a ×40 dry lens (0.75 numerical aperture). The second step should have been to run an immunocytochemical protocol to identify the neurons using a neuronal protein marker (anti-NeuN rabbit Antibody, ABN78, dilution 1:100; Merck). However, due to fungal contamination of the samples during storage, it was not possible to run the second step of the technique to estimate neuronal numbers, and we only obtained reliable numbers on total cell numbers. All cell counts were done blindly regarding the identity of the samples.