Cls3464

CCR7-dependent migration of M1-macrophages towards CCL19 was measured using a Boyden chamber assay. Briefly, 200 μL of M1-macrophages in suspension (5 × 10⁵ cells/mL), previously incubated with CAF-CM during 48 h (as described above), was added to the top chamber of Transwell culture inserts (Ø = 6.5 mm, pore size 8 μm, Cat. # CLS3464, Sigma-Aldrich, St. Louis, MO, USA). Bottom chambers were filled with fresh standard fibroblast growth medium (600 μL) or with CM from irradiated and non-irradiated CAF cultures, in the presence or absence of CCL19 (50 ng/mL) (Cat. # 130-105-744, Miltenyi Biotec, Bergisch Gladbach, Germany) and placed in the upper compartment of a Transwell Plate. After incubation in a humidified atmosphere (5% CO₂, 37 °C, 3 h), macrophages that had migrated into the lower compartment were harvested and counted under light microscopy.

For invasion assay, we used modified two-chamber plates with an 8-μm pore size (Cat#. CLS3464, Sigma-Aldrich, USA) coated with 1 mg/ml matrigel (Cat#. E1270, Sigma-Aldrich, USA). GPR56 control and knockdown cells (10⁴ cells) were added in serum-free media onto the top chamber. In the lower chamber, complete media was used as a chemoattractant. After incubating for 24 hours, the non-invading upper chamber cells were removed by gentle wiping with a cotton-tipped swab. Invading cells on the lower surface of the membrane were fixed with 4% formaldehyde in PBS for 10min, permeabilized with methanol for 20min, stained with 0.4% crystal violet (Cat#. V5265, Sigma-Aldrich, USA) for 15min, counted, and photographed at a magnification of 10X. The fold increase in invasion was calculated by dividing the total number of invading cells from the GPR56 knockdown cells group by those from the GPR56 control group.