Thunder dmi8 3d fluorescence imaging system

U-2 OS cells were cultured and plated in a 6-well plate (Celltreat, Pepperell, MA, USA). Once adherent, cells were treated with 5 μg/mL of 0.1 μm PS-MPs or 20 μg/mL of 2 μm PS-MPs for 24 h. After exposure, the cells were washed with sterile PBS (Gibco, Waltham, MA, USA), detached, centrifuged in DMEM (Gibco, Waltham, MA, USA) at 1000× g at 4 °C for 5 min to remove excess PS-MPs, and re-plated on 13 mm coverslips in a 6-well plate. After 24 h, the cells were washed 3 times with PBS, fixed for 10 min with 4% formaldehyde at RT in PBS, permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MO, USA) in PBS at RT for 15 min, stained with Hoescht (1:2000, H1399, Invitrogen, Waltham, MA, USA) and Phalloidin (1:500, PF7501, EPM Scientific, New York, NY, USA) for 5 min at RT, washed 3 times in PBS, and mounted onto glass slides with an aqueous mounting medium. Fluorescence imaging (Leica THUNDER DMi8 3D Fluorescence Imaging System, Leica Biosystems, Wetzlar, Germany and LAS X 3D Analysis Software v. 2018.7.3, Leica Biosystems, Wetzlar, Germany) was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.

For detection of MPs, representative post-fixed brain, lung, heart, liver, kidney, GI, and spleen samples from control and high-dose groups from both young and old cohorts were examined. Frozen embedded tissues were sectioned at 20 μm using a cryostat (Leica BioSystems, CM1950, Wetzlar, Germany) taken at −21 °C and collected onto glass slides (VWR Colorfrost^® Plus, Radnor, PA, USA). The sections were allowed to dry at 30 °C for 5–10 min before being circled using a wax pen. The sections were washed for 5 min in TBS, permeabilized with 0.3% Triton X-100 (Sigma, St. Louis, MO, USA) in TBS for 30 min, and stained with DAPI (1:5000, 5.08741.0001, MilliporeSigma, Burlington, MA, USA) for 10 min. Sections were washed again in TBS for 5 min and coverslipped (VWR, Radnor, PA, USA) with aqueous anti-fading mounting medium (20 mM Tris pH 8.0, 0.5% N-propyl gallate, 50% glycerol). Fluorescence imaging (Leica THUNDER DMi8 3D Fluorescence Imaging System, Leica Biosystems, Wetzlar, Germany and LAS X 3D Analysis Software v. 2018.7.3, Leica Biosystems, Wetzlar, Germany) was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.

Fluorescence imaging was used to evaluate and document MPs and the immunolabeling results (Leica THUNDER DMi8 3D Fluorescence Imaging System, Leica Biosystems, Wetzlar, Germany, and LAS X 3D Analysis Software v. 2018.7.3, Leica Biosystems, Wetzlar, Germany). Images were processed using the appropriate software (FIJI v2.1.0/1.53c, Madison, WI, USA) [52 (link)].