Cell discoverer 7 high content imaging system

A. muciniphila were stained using pHrodo Red Dye (Invitrogen, Thermo Fisher Scientific) as per the manufacturer’s recommendation, then incubated in RPMI 1640 containing 1% FCS and 1 nM human recombinant ITLN1 (R&D Systems, Bio-Techne Ltd.) or 1% FCS-containing RPMI 1640 alone, for 2 h at 37°C.
Monocyte-derived macrophages on 96-well plates (4 × 10⁴ cells/well) were stained with Hoechst 33342 (Thermo Fisher Scientific) for 1 h at 37°C for cell nucleus visualization and washed with RPMI-1640. Labeled and ITLN1-coated or uncoated A. muciniphila (ratio 50:1 of bacterial cells:macrophages) were added to macrophages. Over time, the increase in pHrodo Red signal (indicating phagocytosis of bacterial cells by macrophages) was quantified using a Zeiss Cell Discoverer 7 high-content imaging system.

Planarian flatworms (Dugesia japonica) were starved 7 days and subjected to post‐ocular head resection. Posterior portions were placed in Planaria water containing MCTR1, MCTR2, or MCTR3 (0.1‐10 nm) or vehicle (0.05% v/v EtOH) for 4 h. In experiments with Rac1 inhibition, posterior portions were placed in Planaria water for 3 h after which Rac1 inhibitor NSC23766 (100 µm) or equal volume DMSO was added to each well for 15 min, followed by MCTR1, MCTR2, or MCTR3 (1 nm) for 45 min. After incubations, Planarian flatworms were transferred into 2% HCl in ddH₂O for 5 min to euthanize animals, followed by 4% paraformaldehyde for 20 min to fix specimens. After washing twice with PBS containing 0.3% Triton X‐100 (PBS+TX‐100), specimens were bleached overnight in 6% H₂O₂ in PBS+TX‐100. Fixed and bleached Planaria were stained for ACs using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore) according to previously published protocols.^[35 (link)
^] Stained samples were mounted on glass microscopy slides with MOWIOL and the number of TUNEL+ cells were determined using the Celldiscoverer 7 high‐content imaging system (Zeiss). Data were analyzed using the ZEN 2.6 software (Zeiss).

Monocyte-derived macrophages were seeded onto 96-well plates at 4 × 10⁴ cells per well and transfected with 1 μM Accell Human Control siRNA or Accell Anti–human GPR101 siRNA SMARTpool for 72 hours in serum-free Accell siRNA Delivery Medium (Dharmacon). Macrophages were stained with Hoechst 33342 (Thermo Fisher Scientific) for 1 hour at 37°C to visualize cell nuclei, washed with RPMI-1640, and incubated with RvD5_n-3DPA (0.001–10 nM) or vehicle (RPMI-1640 containing 0.1% ethanol) for 15 minutes at 37°C. pHrodo Green–labeled S. aureus bioparticle conjugates (Invitrogen, Thermo Fisher Scientific) were opsonized by incubation in PBS containing 20% (v/v) human serum. Opsonized pHrodo Green–labeled S. aureus bioparticles were directly added to the macrophages at a final concentration of 5 μg per well, and the increase in pHrodo Green signal over time (representing phagocytosis of bacterial particles by macrophages) was quantified using a Zeiss Celldiscoverer 7 high-content imaging system.