Reagents

• CD4+ T cell Isolation Kit, Miltenyi Biotec, #130-055-101, $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 80 µl buffer per 10⁷ cells (for fewer cells use same volume).
4. Add 20 µl Hapten-Antibody Cocktail per 10⁷ cells, mix well.
5. Incubate 10 minutes at 6-12°C (refrigerator door).
6. Wash cells with 10-20x the labeling volume of buffer. Centrifuge 300xg for 10 minutes. Remove supernatant.
7. Repeat this washing step.
8. Resuspend cell pellet carefully in 80 µl buffer per 10⁷ total cells.
9. Add 20 µl MACS Anti-Hapten MicroBeads per 10⁷ total cells, mix well.
10. Incubate 15 minutes at 6-12°C (refrigerator door).
11. Wash cells with 10-20x the labeling volume of buffer.
12. Centrifuge 300xg for 10 minutes. Remove supernatant and resuspend cells in buffer (500 µl per 10⁸ total cells). Keep cells on ice.
13. Place column in separation magnet on multistand (one LS column will hold 10⁸ positively labeled cells). Place 15 ml tube below column.
14. Wash column with 3 ml buffer, discard effluent. Replace 15 ml tube.
15. Add magnetically labeled cell-suspension on top of column (max. 10 ml).
16. Allow unlabeled cells to pass through drop by drop.
17. Collect effluent as negative fraction (CD4⁺ T cells) in 15 ml tube.
18. Wash column 4 times with 3 ml buffer, collect effluent in same negative fraction 15 ml tube. Keep cells on ice.
19. Remove column from separator magnet and place over clean 15 ml tube.
20. Pipette 5 ml buffer on top of column and flush out labeled cells (non-CD4⁺ T cell fraction) using the plunger. Keep cells on ice.
21. Count both fractions.
22. Check purity on FACS.