Raw 264.7 cells were cultured in complete growth medium (DMEM + 10% FBS + 1% Penicillin/Streptomycin) at 37 °C with 5% CO2. One day before the experiment, cells were seeded onto sterile coverslips in a 6-well plate at a density of 4 × 105 cells/mL and incubated overnight. Then cells were treated with or without LPS (100 ng/mL) for 24 h in serum-free media.
For immunofluorescence staining,37 (link) cells on coverslips were fixed with 4% paraformaldehyde for 30 min. Goat anti-mouse IL-13Rα2 antibody (1:5000) was used to detect IL-13Rα2, and followed by incubation with Alexa Fluo 594 chicken anti-goat secondary antibody, (1:5000, Invitrogen, Waltham, MA) for 30 min. IgG control was performed excluding the primary antibody incubation. Cell nuclei were countered stained with DAPI.
For probe binding experiments, cells treated with or without LPS were fixed with 4% paraformaldehyde for 30 min and incubated with 1 μM IL-13-functionalized Gd3N@C80 MRI probe labeled with TAMRA and washed with PBS twice. Cell nuclei were counterstained with DAPI. Fluorescence images were taken with a fluorescence microscope (LSM 510-UV, Carl Zeiss, Germany) and processed by NIS Element Basic Research software (Nikon Instruments Melville, NY) with the exact same setting for infected and sham tibia samples following our published protocols.38 (link)
For immunofluorescence staining,37 (link) cells on coverslips were fixed with 4% paraformaldehyde for 30 min. Goat anti-mouse IL-13Rα2 antibody (1:5000) was used to detect IL-13Rα2, and followed by incubation with Alexa Fluo 594 chicken anti-goat secondary antibody, (1:5000, Invitrogen, Waltham, MA) for 30 min. IgG control was performed excluding the primary antibody incubation. Cell nuclei were countered stained with DAPI.
For probe binding experiments, cells treated with or without LPS were fixed with 4% paraformaldehyde for 30 min and incubated with 1 μM IL-13-functionalized Gd3N@C80 MRI probe labeled with TAMRA and washed with PBS twice. Cell nuclei were counterstained with DAPI. Fluorescence images were taken with a fluorescence microscope (LSM 510-UV, Carl Zeiss, Germany) and processed by NIS Element Basic Research software (Nikon Instruments Melville, NY) with the exact same setting for infected and sham tibia samples following our published protocols.38 (link)