Laser scanning microscope 780

Spleens were fixed overnight in medium containing 0.05 M phosphate buffer, 0.1 M l-lysine, pH 7.4, 2 mg/ml NaIO4, and 10 mg/ml paraformaldehyde and were dehydrated with 30% sucrose in phosphate buffer. Tissues were frozen in Tissue-Tek O.C.T. compound (Sakura Finetek) and sectioned using a CM1850 cryostat (Leica). 20-μm frozen sections were permeabilized with 1% Triton X-100 (Sigma) for ten minutes at room temperature, blocked for 30 minutes with Background Buster (Innovex Biosciences), stained over night at 4 °C in a humidified chamber with an antibody cocktail (Supplemental Table 2), then mounted with Immu-Mount (Thermo Sceintific). Immunofluorescence confocal microscopy was performed with the Zeiss 780 laser scanning microscope (Carl Zeiss; air objective 20× Plan-Apochromat with NA 0.5) using multichannel frame scans. The ZEN 2012 software (Carl Zeiss) was used for image acquisition. For half spleen images, the ZEN 2012 tile scan function was used to stitch individual 20x images together. Quantification of GC area, GC T_FH cells and image processing was performed using Imaris 8.1 software (BITPLANE).

HeLa cells were seeded and cultured in 35 mm glass-bottom dishes overnight. Cells were first washed with phosphate-buffered saline (PBS, pH 7.4) 3 times. As a result of this treatment, cells are in a neutral extracellular environment (around pH 7.4). An amount of 2.5 µL of FNDs (1 mg/mL stock concentration) were mixed with 25 µL pHrodo Green Dextran in 500 µL DMEM high glucose (4500 mg/L) and added to cells. Then we incubated for 30 min, removed the medium, and washed cells with PBS (pH 7.5) 3 times. Finally, after 6 h of incubation, a lysosome tracker (LysoView 405) was added for live-cell imaging. Z-stack confocal imaging was performed with a Zeiss 780 laser-scanning microscope (Zeiss, Jena, Germany). Nanodiamonds were detected at 561/659 nm, pHrodo Green Dextran at 509/533 nm, and LysoView 405 at 400/464 nm.

Cells were seeded (100,000
cells/mL) in 35 mm glass-bottom cell culture dishes (Greiner bio-one)
and incubated with 10 μg/mL of FNDs for 5, 10, 15, 20, and 25
h, respectively. Then, cells were fixed with 4% PFA for 10 min and
stained with DAPI and fluorescein phalloidin (FITC- phalloidin for
staining F-actin). Confocal images were acquired with a Zeiss 780
laser-scanning microscope (Zeiss, Jena, Germany). FNDs were detected
at ex/em = 561/659 nm; DAPI and FITC were imaged at 358/461 nm and
495/510 nm, respectively.^{34 (link)}Diffraction
PSF 3D and iterative deconvolve 3D plugins of FIJI were used to improve
the signal-to-noise ratio before counting.
For each cell type
(HEK-WT, HEK PQ, HEK PQi), around 100 random
cells were selected and analyzed. Z-stack confocal images containing
the whole cell volume were acquired and deconvolved. The numbers of
FNDs per cell were then analyzed using the 3D object counter plugin
of FIJI. A size filter was set to 8 and the threshold was set to 40.
This number was determined earlier from a control group to separate
the FND signal from the background. This was the smallest number,
where the signal from the control group was 0.