70 μm mesh

Thymuses from WT or Yap-cKO mice (8–10 weeks old) were mechanically disrupted by being pushed through a 70 μm mesh (Falcon) with an insulin syringe plunger and washed with PBS. Cells were treated with ACK red blood cell lysis buffer, and thymocyte single-cell suspensions were stained for analysis by flow cytometry as described earlier. Cells were stained with dead cell dye and antibodies for the following surface markers: CD3 BUV737 (BD), CD4 BUV395 (BD), CD8 PerCP-Cy5.5 (Biolegend), TCRβ BV510 (Biolegend), CCR7 PECy7 (Biolegend), H-2Kb PE (Biolegend), CD69 BV421 (Biolegend), CD45R/B220 APCFire750 (Biolegend), CD25 APCFire750 (Biolegend), GL3 APCFire750 (Biolegend), and NK1.1 APCFire750 (Biolegend). Thymocytes were also stained intracellularly with anti-Nur77 APC (BD) using the eBioscience Foxp3/Transcription factor staining buffer set, as described earlier.

CAFs and primary colorectal NFs were isolated from breast cancer tissues and adjacent normal tissue. Briefly, the specimen was cut into sections, then subjected to collagenase IV (Invitrogen, CA, United States) digestion for 3 h at 37°C. After digestion, it was passed through a 70 μm mesh (BD Falcon, CA, United States). We centrifuged the filtrate and cultivated the cells in red blood cell lysis buffer to eliminate red blood cells, washed the remaining cells with PBS. The cells for analysis were re-suspended in FACS staining buffer for flow cytometry, cells for culturing were then plated in DMEM with low glucose content containing 10% FBS (Gibco, United States) and 1% penicillin/streptomycin, adherent cells were removed to other culture dishes for the cultivation of CAFs and NFs. For the collection of CM, 2 × 10⁶ cells were seeded onto a 10-cm plate and cultured for 36 h, the medium was replaced with 5 mL fresh F12 without serum and cells were cultured for a further 24 h, the CM was collected and filtered and stored at −40°C.

The Auditory Cortex (AC) tissues of different ages were dissected, then subjected to 2 mg/ml Papain (Sigma, USA) and DNase digestion for 30min at 37°C. After digestion, it was passed through a 70 μm mesh (BD Falcon, CA, USA). We centrifuged the filtrate and washed the remaining cells with PBS. The cells for analysis were re-suspended in FACS staining buffer for flow cytometry. Cells were stained with anti-CaV1.3 (1:50) for 1 h, and washed 3 times with PBS, then stained with anti-rabbit, APC (1:1000; Cell Signaling Technology, USA) and anti-NeuN, FITC (1:100; Cell Signaling Technology, USA) for 30min at 37°C. Labeled cells were analyzed on BD FACSVerse Flow Cytometer, and the data were processed using FLOWJO software (Treestar).

B16 tumors from WT and Yap-cKO mice were dissected, mechanically disrupted, and digested in serum-free media containing 2 mg/ml collagenase type I (Worthington) and DNase I (Sigma) for 30 minutes at 37°C in a rotator. Tumor digests were then passed through a 70 μm mesh (Falcon) using an insulin syringe plunger and washed with PBS. Cells were treated with ACK red blood cell lysis buffer (Gibco), and tumor single-cell suspensions were prepared for staining and analysis by flow cytometry, using DAPI (Biolegend) and antibodies for CD45 BV510 (Biolegend), CD3 BUV737 (BD), CD4 BUV395 (BD), and CD8 PerCP-Cy5.5 (Biolegend). For determining absolute numbers of tumor-infiltrating T cells by flow cytometry, AccuCount fluorescent particles (Spherotech) were added to the tumor digests.

Freshly obtained adipose tissue samples were dissociated by being cut into small pieces. Stromal vascular cells were isolated from 1 g minced tissues using collagenase (C6885, Sigma-Aldrich, Buchs, Switzerland; 1.3 mg/ mL DMEM/ g adipose tissue) in DMEM on a shaker at 37 °C. After the one-hour digestion, the cell suspension was filtered through a 70-μm-mesh (BD Biosciences, Allschwil, Switzerland) in order to remove undigested tissue. The filtered cell suspension was centrifuged at 300 g for 10 min. The floating adipocyte fraction including the supernatant was collected and centrifuged again for 10 min at 300 g. After the second spin, the two pellets were combined and resuspended into 10 mL of 4 °C cold FACS buffer (PBS containing 0.5% bovine serum albumin and 2 mM EDTA). Following an additional washing step, cells were counted and then diluted to a final concentration of 1 × 10⁷ cells/ mL. Care was taken during specimen handling to ensure that cell isolation was as consistent as possible. As much tissue as possible was analyzed to maximize the number of cells isolated; however, in some individual samples, the number of cells isolated imposed limitations on the number of CD makers that could be studied with flow cytometry.