Reagents

• MidiMacs Separation Unit, Miltenyi Biotec, #130-042-302, $___
• MultiMacs Multistand, Miltenyi Biotec, #130-042-303, $___
• LS Columns, Miltenyi Biotec, #130-042-401, $325 / 25pk
• Dulbecco's Phosphate Buffered Saline (PBS),calcium/magnesium free, BioWhittaker, #17-515F, $___
• Bovine Serum Albumin (BSA), Sigma, #A2153, $___
• EDTA, 0.5 M, Sigma, #E7889, $___
• Separation & Wash Buffer: 500 ml PBS, 2.5 g BSA, 2 ml 0.5 M EDTA
• Antigen specific microbeads, Miltenyi Biotec Inc, 2ml, $495*

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 MACS microbeads per 10⁷ cells, mix well.
5. Incubate 15 minutes at 6-12°C (refrigerator door).
6. Optional: Add specific fluorochrome conjugated antibody and incubate additional 5-10 minutes to evaluate separation efficiency by FACS.
7. Wash cells with 10-20x the labeling volume of buffer.
8. Centrifuge 300x g for 10 minutes. Remove supernatant and resuspend cells in buffer (500 µl per 10⁸ total cells). Keep cells on ice.
9. Place column in separation magnet on multistand (one LS column will hold 10⁸ positively labeled cells). Place 15 ml tube below column.
10. Wash column with 3 ml buffer, discard effluent. Replace 15 ml tube.
11. Add magnetically labeled cell-suspension on top of column (max. 10 ml).
12. Allow negative cells to pass through drop by drop.
13. Collect effluent as negative fraction in 15 ml tube.
14. Wash column 3 times with 3 ml buffer, collect effluent in same negative fraction 15 ml tube. Keep cells on ice.
15. Remove column from separator magnet and place over clean 15 ml tube.
16. Pipette 5 ml buffer on top of column and flush out labeled cells (positive fraction) using the plunger. Keep cells on ice.
17. Count both fractions.
18. Check purity on FACS.