Pe cy7 anti mouse podoplanin

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).

Renal tissue was minced, digested by collagenase, washed with PBS, and finally prepared as a single cell suspension. The cells were then treated at 4 °C for 30 min in the dark with the following antibodies: APC anti-mouse CD11b (BioLegend, USA, 1:100), PE anti-mouse CD86 (BioLegend, USA, 1:50), FITC anti-mouse MHC-II (BioLegend, USA, 1:50), Percp/cy5.5 anti-mouse F4/80 (BioLegend, USA, 1:50), PE-cy7 anti-mouse Podoplanin (BioLegend, USA, 1:50). Flow cytometric analysis was performed using a BD FACSCanto II (BD, USA) and FlowJo software.