CD31 MicroBead Kit Purification

Note: Volumes are given for 1X 107 change the volume according to the cell number

MAGNETIC LABELING:

1. Count the number of cells and take 1x107 – more if we want2. Centrifuge cells at 300xg for 3 min and aspirate supernatant completely.3. Resuspend cells in 60 ul of medium (maximum concentration of 1x107)4. Add 20 ul of FcR blocking reagent. Vortex briefly or resuspend gently.5. Add 20 ul of CD31 microbeads to the mixture.6. Incubate 15 min at 40 C. (Keep under sterile condition)7. Add 1 ml of medium and centrifuge cells at 300xg for 3 min.

(add gently(pipette up and down 3 times) and don’t resuspend )8. Resuspend cell pellet in 1 ml of medium.

MAGNETIC SEPARATION:

1. Place LS column in the magnetic field of Midimacs separator.2. Prepare column by rinsing with 3 ml of medium.3. Apply cell suspension onto the column.4. Collect unlabeled cells which pass through and wash column with 3x3 ml of medium.

Perform washing steps by adding medium three times. (Only add new medium when the column reservoir is empty. Collect total effluent: this is unlabeled fraction)

5. Remove column from the separator and place it on a suitable collection tube.6. Pipette 5 ml of medium onto the column. Immediately flush out the magnetically labeled cells

by firmly pushing the plunger into the column.

7. Cells can be directly analyzed by flow cytometry for purity or taken into culture.(cells should lbe seeded at a density of approx. 150,000 per T-75 flask.)Note: Should fibroblast contamination outgrow endothelial cells upon prolonged cultivation. Endothelial cells should be re-purified using CD31 microbeads.