Hello,I was wondering if you could tell me approximately how...

Hello,

I find the very last steps somewhat confusing:
"4) Rotate bead-pellet at 4°C for 30 minutes. Place tube on magnet for 2-3minutes, remove supernatant, and resuspend beads in 2ml of M199. Repeat for a total of four washes.
5) To liberate cells from anti-CD31 antibody coated beads, add 5mM EDTA in PBS and rotate at37°C for 10 minutes.
6) After 4 washes, place the beads in culture media. For culture of mouse EC, coat the plate with 0.2% gelatin for 20 minutes prior to plating cells."

After washing the beads four times in step (4), you are liberating the cells with 5mM EDTA, so I assume the cells are now in the supernatant, and no longer attached to the beads. So why do you wash the beads again four more times in step (6) and then plate the beads in the culture medium?

Thank you.

Hi,

Normally you could get aorund 10000~50000 endothelial cells from 5 mice in 5-6 weeks old. This is we roughly got in the experiment, but a lot of cells are lost during the isolation and purification precedure.