Legacy moflo mls high speed cell sorter

The eyes were enucleated from postnatal day 4–7 mice and transferred to ice-cold 1 × HBSS. The eyes were dissected and retinal dissociation was performed using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA) according to the manufacturers' instruction. The cells were re-suspended in 1% FCS in phosphate-buffered saline (PBS) at a concentration of 20 × 10⁶ cells ml⁻¹. Propidium iodide (1 μg ml⁻¹, Sigma-Aldrich) was added before FACS sorting to exclude dead cells. GFP-positive photoreceptors were sorted using the Beckman Coulter Legacy MoFlo MLS High Speed Cell Sorter or Beckman Coulter MoFlo XPD cell sorter. Different emission filters and lasers were used for GFP (529/28 emission filter; 488 laser) and Propidium iodide (625/26 filter; 561 laser). The sorted cells were collected in EBSS containing 10% fetal bovine serum and kept on ice. The cells were spun at 100 RCF for 20 min and the cell pellet was resuspended at a concentration of 200,000 cells μl⁻¹ in EBSS containing 0.005% DNase I and kept on ice before transplantation.

Primary LMVECs were isolated as previously described (76 (link)). Briefly, lungs from female C57BL/6 mice were dissected, digested in a 0.5-mg/ml collagenase solution for 1 hour at 37°C, and filtered through a 70-μm cell strainer (Thermo Fisher Scientific). Suspended cells were washed, blocked with 10 μg/ml murine IgG (I5381, Sigma-Aldrich), and stained with isolectin-B4–FITC (L2895, Sigma-Aldrich), anti-CD31–PE (102408, BioLegend), and anti-CD105–APC (120414, BioLegend) antibodies, diluted to 1 μg/ml in PBS with 2.5% FBS. Isolectin-B4⁺CD31⁺CD105⁺ cells were sorted using a Beckman Coulter Legacy MoFlo MLS High Speed Cell Sorter and cultured.