Isolation and Characterization of Lymphatic Endothelial Cells

After generating a single cell suspension and staining for cpLECs with Ghost UV450 (Tonbo Biosciences, Catalog #: 13-0868-T500), CD31 Alexa 647 (BD Biosciences, Catalog #: 565608), Podoplanin PE (eBioscience, Catalog #: 12-5381-82), and CD45 APC-Cy7 (BioLegend, Catalog #: 103116), the cells were sorted with a FACS Aria III with a nozzle size of 130 μm at the UW Flow Core satellite facility in the UW Biotechnology Center. LECs were gated for singlets using both FSC and SSC, dead cells excluded by gating for Ghost^negative live cells, and LECs were identified using both CD31⁺ Podoplanin⁺ after excluding both CD45^intermediate microglia and CD45⁺ leukocytes as previously described^{2 (link),6 (link),8 (link),19} . Cells were sorted into either FACS buffer (1% BSA in PBS) for scRNA-seq, or RPMI containing 10% FBS, 2-mercaptoethanol, and penicillin/streptomycin for in vitro co-culture experiments.