Fitc anti mouse ter 119

To sort PDGFRβ⁺ IntSCs or intestinal LECs, the enriched fractions went on RBC lysis by suspension in ACK lysis buffer (Gibco) for 5 min at RT. After blocking Fcγ receptors with mouse anti-CD16/CD32 (553141, BD Bioscience), cells were incubated for 15 min with indicated antibodies in FACS buffer (2% FBS in PBS). After several washes, cells were analyzed by FACS Canto II (BD Biosciences) and the acquired data were further evaluated by using FlowJo software (Treestar). Cell sorting was performed with FACS Aria Fusion (Beckton Dickinson). Dead cells were excluded using DAPI (Sigma-Aldrich) staining and cell doublets were systematically excluded. Fluorescence intensity is expressed in arbitrary units on a logarithmic scale, and forward scatter and side scatter are represented on a linear scale. The following antibodies were used for flow cytometry: FITC anti-mouse CD45 (11-0451-85, rat monoclonal, Biolegend); FITC anti-mouse TER-119 (11-5921-85, rat monoclonal, Biolegend); APC anti-mouse CD31 (551262, rat monoclonal, BD Bioscience); and PE/Cy7 anti-mouse Podoplanin (127412, syrian hamster monoclonal, Biolegend).

To sort dura mater or brain ECs, CD45⁺ hematopoietic or CD45⁻ non-hematopoietic cells by FACS, tissues were isolated as described above and dissociated in collagenase type I (1 mg/ml, Worthington), dispase (1 mg/ml, Gibco), and DNase I (0.1 mg/ml, Merck) dissolved in DMEM/F12 supplemented with 5% fetal bovine serum (FBS) at 37 °C for 30 min. Cell suspensions went on RBC lysis by suspension in ACK lysis buffer (Gibco) for 5 min on ice and blocked with mouse anti-CD16/CD32 (553141, BD Bioscience) before being incubated for 20 min with indicated antibodies in FACS buffer (2% FBS in PBS). After several washes, brain ECs were enriched using automatic magnetic-associated cell sorting (MACS, Miltenyi-Biotec) prior to be being stained with fluorochrome-conjugated primary antibodies. Cell sorting was performed with FACS Aria II (Beckton Dickinson). Dead cells were excluded using DAPI (Sigma-Aldrich) staining and cell doublets were systematically excluded. The following antibodies were used for MACS: biotin anti-mouse CD31 (rat monoclonal, 130-119-562, Miltenyi-Biotec); anti-biotin microbeads (130-090-485). The following antibodies were used for FACS: PE anti-mouse CD31 (rat monoclonal, 102508, Biolegend); APC anti-mouse CD45 (rat monoclonal, 103112, Biolegend); FITC anti-mouse TER-119 (rat monoclonal, 116206, Biolegend).