Isolation of lymphatic endothelial cells

                          Dermal Cell Suspensions           
1. Dermatomed 0.8-mm split-thickness skin was obtained from adult healthy individuals undergoing elective surgery.
2. Dermal sheets were prepared by incubation of split-thickness skin with dispase (50 U/ml) for 30 min at 37°C, and subsequent removal of the epidermis.
3. Dermal cells were released from the tissue by scraping.
4. Cells were pelleted, resuspended in primary EC growth medium, and either seeded on fibronectin (10 μg/ml)-coated dishes for in vitro expansion, or subjected to immunostaining as described below.

Immunoisolation of EC Subsets from Dermal Cell Suspensions
1. The procedures below are for the isolation of podoplanin⁺ and podoplanin⁻ microvascular ECs from freshly prepared dermal cell suspensions.
2. Dermal cells were incubated for 7 min at 37°C in trypsin/EDTA.
3. Cell pellets were resuspended in ice-cold PBS/2 mM EDTA/0.5% BSA, and the resulting cell suspension was filtered through a sterile sieve with 200 μm mesh-size, to remove fibers and cell aggregates.
4. Cells were adjusted to 10⁷ cells/ml in primary EC growth medium, supplemented with 10 μg/ml normal goat IgG, and exposed simultaneously to rabbit anti-podoplanin serum (final concentration: 1:100), anti–CD34-PE and anti–CD45-RPE-Cy5 (2 μg/ml each), or to appropriate control Abs for 45 min on ice.
5. After two washes, the binding of rabbit Abs was revealed by incubation with goat anti–mouse F(ab′)2 FITC (10 μg/ml for 30 min).
6. The staining procedure did not interfere with cell viability, as determined by trypan blue exclusion.
7. Cells were washed, resuspended in cold PBS/2 mM EDTA/0.5% BSA, and subjected to flow cytometry analysis.
8. Podoplanin⁺CD34⁺CD45⁻ and podoplanin⁻CD34⁺ CD45⁻ EC subsets were sorted on a FACStarPLUS™ flow cytometer.
9. The purity of the sorted cell fractions was analyzed on a FACScan™ and always exceeded 98%.
10. 0.5 × 10⁶ sorted cells/ml were cultured on fibronectin (10 μg/ml)-coated 96-well flat bottom plates in primary EC growth medium.

Isolation of Subsets from Bulk Cultures of Dermal ECs
1. Freshly isolated dermal cell suspensions were cultured in primary EC growth mediu until confluent monolayers were formed.
2. Loosely attached cells were discarded and adherent cells harvested by trypsinization as described above.
3. 108 streptavidin-conjugated paramagnetic beads were coated with 10 μg biotinylated UEA I.
4. Dermal cells (10⁷/ml) were incubated with UEA I–coated beads (bead/cell ratio, 1:4), and ECs attached to the beads were isolated using a magnet.
5. UEA I+ ECs were passaged twice, harvested by trypsinization, and exposed simultaneously to anti-podoplanin (1:100), anti-CD31–biotin, and anti-CD45–RPE-Cy5 Abs (2 μg/ml each) followed by streptavidin-PE and goat anti–mouse F(ab′)2 FITC.
6. ECs were sorted into podoplanin⁺CD31⁺CD45⁻ and podoplanin⁻CD31⁺CD45⁻ subsets on a FACStar^PLUS™.