Lionheart lx

RAW264.7 macrophages were fixed with 4% paraformaldehyde for 15 min and permeated with 0.3% Triton X-100 for 15 min at room temperature. After washed with PBS for 1 h, the cells were incubated with primary antibodies against hnRNPK, FLIP and NLRP3 (Abcam) at 4 °C overnight. The coverslips were exposed to Alexa Fluor conjugated-secondary antibodies (Cell Signaling Technology) for 1 h in the dark. The nucleus was marked with 4′, 6-Diamidino-2-phenylindole (DAPI). The stained images were observed using an Inverted/Fluorescence Microscope (LIONHEART ^LX, BioTek).

After euthanasia, the subcutaneous implants were fixed with 10% formalin. Following fixation, the implants were washed and placed into a processor that dehydrated the samples in 70% alcohol, followed by 95%, 100%, and xylene. The samples were then embedded in paraffin and cut into slices of 5 microns using a microtome (Accu-Cut SRM 200 Rotary Microtomoe, Sakura Finetek USA, CA). Slides were stained with hematoxylin and eosin (H&E) and Goldner’s trichrome (Sigma-Aldrich). Images were obtained with Lionheart LX (Biotek Instruments Inc, Winooski, VT) at 4× and captured using Gen 5 software.

After euthanasia, at 4 weeks post-surgery, the subcutaneous implants were fixed with 10% formalin. Following fixation, the implants were washed and placed into a processor which dehydrated the samples sequentially in 70%, 95% and 100% alcohol, followed by xylene. The samples were then embedded in paraffin and cut into slices of 5 microns using a microtome (Accu-Cut SRM 200 Rotary Microtomoe, Sakura Finetek USA, Torrance, CA, USA). Slides were stained with Hematoxylin and Eosin (H&E) and Goldner’s trichrome (Sigma-Aldrich). Images were obtained with Lionheart LX (Biotek Instruments Inc., Winooski, VT, USA) at 4× and captured using Gen 5 software (Biotek Instruments Inc., Winooski, VT, USA)).

CAFs were fixed with 4% paraformaldehyde (PFA, Cat. No.158,127; MERCK/Sigma-Aldrich, Darmstadt, Germany) for 30 min and then permeabilized with Triton X-100 (0.01%, Thermo Fisher Scientific, MA, USA) for 10 min. After blocking with 0.1% bovine serum albumin (BSA), the cells were incubated with primary antibody overnight at 4°C. The primary antibodies were as follows: FAP (1:100, #66,562, Cell Signaling Technology/CST, MA, USA), Vimentin (1:150, #5741, CST, MA, USA), and α-SMA (1:100, #19,245, CST, MA, USA). After washing, the samples were incubated with goat anti-rabbit IgG (Alexa Fluor® 488, ab150077, Cambridge, UK) for 30 min. The cells were counterstained with DAPI (1 mg/mL, Sigma-Aldrich, Missouri, USA) and blocked with glycerin. Staining was visualized using a fluorescence microscope (Lionheart LX, BioTeK, Vermont, USA).

MCF-7 spheroid uptake experiments
were imaged with a Lionheart LX Automated Microscope (BioTek, Winooski,
VT). The Lionheart LX microscope has 4 LED/filter cubes corresponding
to DAPI, GFP, Texas Red, and Cy7 and is controlled using BioTek’s
Gen 5 software. Confocal microscopy for LIVE/DEAD imaging was performed
by using a Molecular Devices ImageXpress Microscope confocal microscope
with a Lumencor light engine as the laser source.

Briefly, BV2 cells were cultured with complete medium overnight for adherence and were stimulated with serum-free medium for 1 h, pre-treated with Exos or Exos-138 (10⁸ particles/mL) for 12 h, and then stimulated with 200 μM H₂O₂ for 6 h in 12 mm glass cell culture chambers (ThermoFisher, Scoresby, VIC, Australia). Then, DCFH-DA was used to stain the cells for 30 min under the conditions of 37°C and 5% CO₂. Intracellular ROS production was determined using a ROS detection kit. The fluorescence intensity was measured using a Cytation 5 Cell Imaging Multimode Reader (BioTek LionheartLX) and analysed using the ImageJ software.
The spinal cords of all groups were collected in vivo and frozen sections were prepared. A frozen section ROS detection kit (BioRab Technology, Beijing, China) was used. The fluorescence was detected at 525 nm using a Nikon Eclipse Ti-SR microscope (Nikon, Japan).