Isolation of mouse lung endothelial cells

Isolation of mouse lung endothelial cells 

Adapted from (Choi, E.Y., et al. Del-1, an endogenous leukocyte-endothelial adhesion inhibitor,

limits inflammatory cell recruitment. Science 322, 1101-1104 (2008).)

Day 1: prep beads

1. 25ul of beads per set of mouse lungs to isolate
2. Wash in PBS
3. Incubate overnight at 4^oC with antibody of choice, alternatively incubate for 1-2 hours at RT

Day 2: Isolatation

1. Remove lungs from mice
2. Wash in 10%FBS/DMEM
3. Move tissue into DMEM only media
4. Mince the tissue using scissors into 1-2mm squares
5. Digest the tissue with collagenase (2mg/ml) at 37^oC for 1 hour, with rocking or shaking
6. Prepare slides/cover slips/6 well plates to incubate cells in.  Collagen or gelatin coat the surfaces (Collagen 50ug/ml in PBS, Gelatin 1mg/ml in PBS)
7. Filter the digested tissue/cells through a 70 um cell strainer, use the plunger of a syringe to smash the last of the tissue to pass through the filter
8. Wash the filter with 2ml of 10%FBS/DMEM to make sure as many cells as possible are collected
9. Centrifuge at 1500rpm to pellet the cells
10. Wash the dynabeads twice using PBS, resuspened in 1ml for every 25ul of beads used 
11. Incubate the cells with dynabeads, 1ml per each set of lungs being isolated from
12. Incubate at RT for 15-20min, rock the mixture during the isolation
13. Recover the bead-bound cells using a magnet
14. Wash twice with 10%FBS/DMEM, after second wash, remove the DMEM mixture and resuspend the cells in the desired volume of EC media in the sterile hood. Transfer the cells onto the growth surface you would like to keep them on and store in the incubator overnight.