TITLE: Dopamine neuron enrichment using MACS
AUTHORS: Tae Wan Kim

[DESCRIPTION]
high yield DA neuron sorting via MACS but using surface marker strategy described recently (Kim et al.,BioRxiv).  

[STEPS]
SECTION: Cell dissociation
1. Use Worthington Papain Dissociation System (LK003150) to dissociate at day 25 of Studer lab dopamine neuron protocol. Add 32 mls of EBSS (vial 1) to the albumin ovomucoid inhibitor mixture (vial 4) and allow the contents to dissolve while preparing the other components. Mix before using and equilibrate within 5% CO2 incubator. Reconstitute for the first use, then store and reuse.
SECTION: Cell dissociation
2. Add 5 mls of EBSS (vial 1) to a papain vial (vial 2). Place vial 2 in a 37°C water bath for ten minutes or until the papain is completely dissolved and the solution appears clear. If solution appears alkaline (red or purple) equilibrate the solution with 95% O2:5%CO2.
SECTION: Cell dissociation
3. Add 500 µls of EBSS to a DNase vial (vial 3). Mix gently -- DNase is sensitive to shear denaturation. Add 250µls of this solution to the vial containing the papain. This preparation contains a final concentration of approximately 20 units/ml papain and 0.005% DNase.
SECTION: Cell dissociation
4. Place 1ml papain solution per 6-well and incubate at 37°C, 5% CO2 for 45 min to 90 min, periodically agitating the plate.
SECTION: Cell dissociation
5. Gently triturate the mixture with 10ml pipette
SECTION: Cell dissociation
6. Carefully remove the cloudy cell suspension, place in sterile screw capped tube and centrifuge at 300g for 5 minutes at room temperature.
SECTION: Cell dissociation
7. Mix 2.7 mls EBSS (vial 1) with 300µls reconstituted albumin-ovomucoid inhibitor solution (vial 4) in a sterile tube. Add 150 µls of DNase solution (vial 3)
SECTION: Cell dissociation
8. Discard supernatant and immediately resuspend cell pellet in DNase dilute albumin-inhibitor solution 
SECTION: Cell dissociation
9. Discard the supernatant and immediately resuspend the pelleted cells in MACS buffer (PBS without Ca2+/Mg2+, 5% BSA, 2mM EDTA) and count cells
SECTION: CD49 negative selection
10. Add CD49e-Biotin (Miltenyi, 130-110-532) at 1in50 dilution and incubate for 5-10 minutes at (2−8 °C). Note: 1) Incubating on ice can require longer staining times, 2) Neuron yield is directly affected by the quality of the differentiation, it is advisable to perform quality control on your cells at day 11 and day 16 to assess % of on-target cells, 3) Yield may also be improved by performing a titration of the antibody
SECTION: CD49 negative selection
11. Wash cells twice by adding 1−2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
SECTION: CD49 negative selection
12. Resuspend cell pellet in 80ul per 10⁷ total cells.
SECTION: CD49 negative selection
13. Add 20 µL of Anti-Biotin MicroBeads (Miltenyi, 130-090-485) per 10⁷ total cells and incubate for 15 minutes at (2−8 °C).
SECTION: CD49 negative selection
14. Wash cells by adding 1−2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
SECTION: CD49 negative selection
15. Resuspend 1.25x10⁸ cells in 500 µL of buffer (for LD columns)
SECTION: CD49 negative selection
16. Place LD Column (Miltenyi, 130-042-901) in the magnetic field of a suitable MACS Separator.
SECTION: CD49 negative selection
17. Prepare column by rinsing with 2 mL of buffer
SECTION: CD49 negative selection
18. Apply cell suspension onto the column.
SECTION: CD49 negative selection
19. Collect unlabeled cells that pass through and wash column with 2×1 mL of buffer.
SECTION: CD49 negative selection
20. Collect total effluent; this is the unlabeled cell fraction (CD49e negative), Perform washing steps by adding buffer two times. Only add new buffer when the column reservoir is empty.
SECTION: CD49 negative selection
21. You may wish to collect CD49e positive fraction (off-target or neural progenitor cells), to do so, add 2x1ml buffer and gently use the provided plunger with the LD columns in a separate tube
SECTION: CD49 negative selection
22. Centrifuge cells 300xg for 10 minutes
SECTION: CD49 negative selection
23. Wash with MACS buffer and centrifuge 300xg for 10 minutes
SECTION: CD184 postivie selection
24. Resuspend cells in 90ul MACS buffer and add 10 µl of a CXCR4-microbead antibody (Miltenyi, 130-100-070) per 10⁷ cells for 10 minutes at (2−8 °C).
SECTION: CD184 postivie selection
25. Scale volumes accordingly
SECTION: CD184 postivie selection
26. Wash cells by adding 1−2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
SECTION: CD184 postivie selection
27. Resuspend up to 10⁸ cells in 500 µL of buffer (LS columns)
SECTION: CD184 postivie selection
28. Place LS column (Miltenyi, 130-042-401) in the magnetic field of a suitable MACS Separator.
SECTION: CD184 postivie selection
29. Prepare column by rinsing with 3 mL MACS buffer
SECTION: CD184 postivie selection
30. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells (CD184 negative).
SECTION: CD184 postivie selection
31. Wash column with the appropriate amount of buffer. Collect unlabeled cells that pass through and combine with the effluent from
SECTION: CD184 postivie selection
32. Note: Perform washing steps by adding buffer aliquots only when the column reservoir is empty.
SECTION: CD184 postivie selection
33. Remove column from the separator and place it on a suitable collection tube.
SECTION: CD184 postivie selection
34. Pipette the 5ml MACS buffer and immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. To increase the purity of magnetically labeled cells, the eluted fraction can be enriched over a second LS Column.
SECTION: CD184 postivie selection
35. Centrifuge cells 300xg for 10 minutes
SECTION: CD184 postivie selection
36. Resuspend cells in 1ml Day 25 media and count
SECTION: CD184 postivie selection
37. Plate 150,000 CD49e negative / CD184 positive cells per cm2 in 4ml Day 25 media (Studer lab dopamine neuron protocol)