Murine Lymphatic Endothelial Cell Purification

To purify urine lymphatic endothelial cells, use cells isolated from the murine dermis after they reach about 80 to 90%Co-fluency, begin by washing pre-coded magnetic beads in a magnetic separator using one milliliter of magnetic bead coating solution. Resuspend the beads in 150 microliters of DMEM containing 0.1%BSA. Next, wash the cells in the 60 millimeter tissue culture plate twice with PBS.

Mix 50 microliters of the antibody conjugated beads with three milliliters of DMEM containing 0.1%BSA and add it to the cells. Seal the dish with biofilm, then incubate the dish on a shaker agitating at 10 RPM at room temperature for exactly 10 minutes. Discard the medium before washing the cells once with PBS.

Quickly inspect the cells to verify the presence of LEC colonies on the plate. Add one milliliter of tripsin EDTA to the cells and incubate for three minutes at 37 degrees Celsius to trypsinize the cells. Inspect the cells for successful cell detachment.

After neutralizing the solution with two milliliters of complete LEC media. Transfer the cell solution to a five milliliter sterile polypropylene tube. Using a magnetic separator, wash the cells with three milliliters of DMEM containing 0.1%BSA to remove unbound cells.

Resuspend the remaining bead bound cells in complete LEC media and plate them onto collagen coated 60 millimeter tissue culture plates. Brightfield and fluorescence imaging easily identified the LYVE1 positive lymphatic endothelial cells after purification and showed several beads attached to them. The cells displayed low-confluency 24 hours after purification, while they were highly cofluent, seven days after purification.