Na plan apochromat oil immersion objective

HeLa-FKBP-VN cells in DMEM culture medium were seeded on either a 2-well ibiTreat slide (ibidi, Germany) or a 6-well plate (Corning, USA) at 50% confluency 12 h before the intracellular protein delivery experiment. MSNs loaded with FRB-VC proteins (MSN-FRB-VCs) in PBS were added to cell culture in a serum free DMEM and incubated with cells at 37 °C for 2 h. Afterwards, the residual particles in the medium were washed out using PBS (1.5 mL per well) followed by a short chloroquine shock (0.5 mM in standard DMEM, 1.5 mL per well in cell culture medium, RT, 5 min) to trigger endosomal protein release. Cells were then incubated in fresh cell culture medium (phenol red free). All the assays (live cell imaging, FACS analysis and fluorescence readout) were performed at 24 h post MSN-FRB-VCs addition. Images were acquired on an UltraVIEW VoX spinning dizc system with laser line combiner (PerkinElmer) assembled with an inverted microscope (Axio Observer D1) using a 63x/1.4 NA Plan-Apochromat oil immersion objective (Zeiss). Images were acquired with an EMCCD camera (C9100-50, Hamamatsu). The microscope was equipped with a humidified and heated environmental chamber set to 37 °C, 5% CO₂ (PeCon). Image acquisition was controlled by the program Volocity (ver. 6.3, PerkinElmer).

Cells were seeded at 7.5 × 10⁴ cells/well on poly-D-lysine–coated 13 mm round coverslips in 24-well plates and maintained at 37 °C in a 5% CO₂ humidified atmosphere. Cells were treated as described previously, then fixed with 4% (w/v) paraformaldehyde in PBS for 10 min at room temperature. Fixed cells were washed 3 × 5 min in TBS, then permeabilized with TBS + 0.1% saponin for 10 min at room temperature. Cells were then blocked for 1 h at room temperature in blocking buffer (TBS, 10% goat serum, and 1% BSA) before incubating with primary antibody (anti-pSer³⁰⁰/pSer³⁰³-hGPR35a, pSer²⁹⁸/pSer³⁰¹-mGPR35 and anti-HA were diluted 1:400 in blocking buffer) overnight at 4 ^oC. Subsequently, cells were washed 3 × 5 min in TBS, then incubated with secondary antibody (Alexa Fluor 488-goat anti-rabbit IgG and Alexa Fluor 488-donkey anti-rat IgG 1:400 dilution in blocking buffer) for 1 h at room temperature. Cells were washed 3 × 5 min in TBS, and coverslips were mounted onto glass slides using VECTASHIELD Mounting Medium with DAPI (Vector laboratories). Images were taken using a Zeiss LSM 880 confocal equipped with a 63x/1.4 NA Plan Apochromat oil-immersion objective.

HEK293T cells were seeded at 0.5 x 10⁵ cells/well on poly-D-lysine-coated 30-mm round coverslips in 6-well plates and incubated for 24 h at 37 °C. Cells were transiently transfected with wildtype or GPR84 mutants each C-terminally tagged with eYFP. After 48 h, cells were washed twice with HBSS and incubated with 100 nM MemBright 640 solution for 10 min at 37 °C. Subsequently, coverslips were placed in a microscope chamber containing HBSS and images were acquired before treatment and 45 min after agonist addition using a Zeiss LSM 880 confocal equipped with a 63x/1.4 NA Plan Apochromat oil-immersion objective. MemBright 640 dye was excited using 633-nm laser light, and emission light was detected over the wavelength range 660 to 700 nm.