Reagents

• Monocyte Isolation Kit, Miltenyi Biotec, #130-053-301, $595

Protocol

1. Isolate PBMC using density gradient centrifugation with Ficoll-Paque.
2. Make sure buffer is cold and degassed before use.
3. Wash cells in buffer and resuspend in 60 µl buffer per 10⁷ cells (for fewer cells use same volume).
4. Add 20 µl FcR Blocking Reagent per 10⁷ cells, mix well.
5. Add 20 µl Hapten-Antibody Cocktail per 10⁷ cells, mix well.
6. Incubate 5 minutes at 6-12°C (refrigerator door).
7. Wash cells with 10-20x the labeling volume of buffer. Centrifuge 300xg for 10 minutes. Remove supernatant.
8. Repeat this washing step.
9. Resuspend cell pellet carefully in 60 µl buffer per 10⁷ total cells.
10. Add 20 µl FcR Blocking Reagent per 10⁷ cells.
11. Add 20 µl MACS Anti-Hapten MicroBeads per 10⁷ total cells, mix well.
12. Incubate 15 minutes at 6-12°C (refrigerator door).
13. Wash cells with 10-20x the labeling volume of buffer.
14. Centrifuge 300xg for 10 minutes. Remove supernatant and resuspend cells in buffer (500 µl per 10⁸ total cells). Keep cells on ice.
15. Place column in separation magnet on multistand (one LS column will hold 10⁸ positively labeled cells). Place 15 ml tube below column.
16. Wash column with 3 ml buffer, discard effluent. Replace 15 ml tube.
17. Add magnetically labeled cell-suspension on top of column (max. 10 ml).
18. Allow unlabeled cells to pass through drop by drop.
19. Collect effluent as negative fraction (enriched monocytes) in 15 ml tube.
20. Wash column 3 times with 3 ml buffer, collect effluent in same negative fraction 15 ml tube. Keep cells on ice.
21. Remove column from separator magnet and place over clean 15 ml tube.
22. Pipette 5 ml buffer on top of column and flush out labeled cells (non-monocytes) using the plunger. Keep cells on ice.
23. Count both fractions.
24. Check purity on FACS.