Cell sorter software

3D tissue was extracted from the gel with Cell Recovery Solution (Corning 354253). Following incubation at 4 °C for 20 min to depolymerize Matrigel, cell spheroids were dissociated with 0.25% trypsin at 37 °C for 10 min. Single cells were obtained by filtering through 40 µm cell strainers (Falcon 2340) after washing with medium containing FBS. Finally, cells were labeled with HLA‐G–PE, ITGA2–PE, or isotype‐matched controls for analysis (Table S5, Supporting Information). Cell viability was analyzed using LIVE/DEAD Fixable Near Red Dead Cell Stain (Life Technologies L10119). The experiments were conducted using SH800S Cell Sorter (Sony Biotechnology). Data were analyzed in Cell Sorter Software (Sony Biotechnology).

All the sorting experiments were carried out using SONY SH800Z Sorter at Biomedicum Helsinki FACS Core Facility, and the data analysis was performed using Sony Cell Sorter software.

Ai14 reporter mice (tdTomato^f/f) were crossed with a tamoxifen-inducible SMACreER recombinase driver line to generate SMACreER:Ai14 double transgenic mice. Leptomeninges of SMACreER:Ai14 mouse pups at ages P0-6 were dissected and digested with 0.25% trypsin. After a 2-day culture, tamoxifen (5 mM) was added to induce red fluorescent protein tdTomato expression in VSMCs. When the mixed primary pial cells (tdTomato⁺ and tdTomato⁻ cells) became 90% confluent, single-cell suspensions were obtained via 0.25% trypsin digestion and maintained in SMCM. tdTomato⁺ VSMCs were sorted with a LE-MA900FP flow cytometer (SONY, Japan). A 100-μm chip (Cat# LE-C3210) and a PBS sheet fluid pressure of 20 psi were used. First, cells were selected with a very wide gating setting using forward scatter area/side scatter area (FSC-A/SSC-A). Second, based on FSC-A/FSC-H (forward scatter high) and further SSC-A/SSC-H (side scatter high), adherent cells were removed from the parental FSC-A/SSC-A gate. Finally, fluorescent events were selected from the nonadherent cells. tdTomato was excited with a 561-nm laser, and its emission was detected with a 585/30 filter. Cells expressing tdTomato were sorted directly into SMCM and seeded on plates for the following primary culture experiments. All FACS data were analyzed using SONY Cell Sorter software.

Flow cytometric evaluation of UDCs was performed using fluorescein isothiocyanate-conjugated anti-human CD90 antibody (BioLegend, #328107) and 7-AAD (BD Biosciences, 51-68981E). UDCs were trypsinized and washed with growth medium, centrifuged at 200 × g for 5 min, collected, and suspended in phosphate-buffered saline (PBS) with 2% FBS. The antibody was added to UDC suspension at an optimal concentration (1:200) and incubated for 30 min on ice in the dark. Moreover, the 7-AAD was added to UDC-suspension at an optimal concentration (1:100) and incubated for 10 min before analysis. The cells were centrifuged, resuspended in PBS with 2% FBS, passed through a 70 μm filter, and analyzed using a SONY FACS SH800S (Sony Biotechnology, San Jose, CA, USA) and Cell Sorter Software (Sony).

SEAM organoids cultured for 12 weeks were analyzed using FACS based on previous reports.¹⁰ (link)^,12 (link) Cells were dissociated using Accutase (Life Technologies) for 30–60 min at 37°C and re-suspended in an ice-cold keratinocyte culture medium (KCM) supplemented with 5% fetal bovine serum (FBS; Life Technologies). The harvested cells were then stained with antibodies against PE-conjugated anti-SSEA4 (MC813-70, BioLegend, San Diego, CA, USA) or PE-conjugated anti-CD317 (BST2, Tetherin) (RS38E, BioLegend), Alexa Fluor 647 (AF647)-conjugated anti-CD104 (ITGB4) (450-9D, BD Biosciences, San Diego, CA, USA) and PE-Cy7 conjugated-anti-CD200 (OX-104, BD Biosciences) for 60 min on ice. After washing twice with phosphate-buffered saline (PBS) (Wako), the stained cells were sorted using an SH800 instrument (SONY Biotechnology, Tokyo, Japan). In all experiments, the harvested cells were stained with isotype antibodies corresponding to each antibody, as in the control experiments (BioLegend and BD Biosciences). Data were analyzed using the Cell Sorter software (SONY Biotechnology).