TITLE: LUHMES (lund human mesencephalic) culturing and differentiation protocol
AUTHORS: Mallory Wright, William J Buchser, ckremitz, jwaligor, bachman@wustl.edu, serenaelia@wustl.edu

[STEPS]
SECTION: LUHMES coating protocol
1.  
0.1 mg/mL
50 µg/µL
Thaw an aliquot of Poly-L-Ornithine solution at room temperature.
SECTION: LUHMES Passaging (~ every 2-3 days)
19. use a 5mL pipette to Triturate cells only 1 or 2 times before seeding.
SECTION: LUHMES Passaging (~ every 2-3 days)
18. Discard the supernatant and resuspend in 1mL LUHMES growth media 
SECTION: LUHMES Passaging (~ every 2-3 days)
17. Transfer cells to 15mL tube and centrifuge for 5 minutes at 1200 RPM
SECTION: LUHMES Passaging (~ every 2-3 days)
16. Neutralize trypsin with 6mL of pre-warmed DMEM/F12
SECTION: LUHMES Passaging (~ every 2-3 days)
15. Add 4mL of the pre-warmed .025% Trypsin/EDTA and place in the incubator for 3 minutes.  


SECTION: LUHMES Passaging (~ every 2-3 days)
13. Remove media and rinse with DPBS
SECTION: LUHMES growth media
12.  
       
 Add recombinant human FGF to media after seeding cells
**See material section for media concentration information**
SECTION: LUHMES coating protocol
6.  
 Storage:  –20 °C in aliquots
1 mg/mL
2 µg/µL
Thaw an aliquot of Fibronectin solution at 5°C .
 

SECTION: LUHMES growth media
11. Change (pre-warmed) media every 1-2 days
 

SECTION: LUHMES coating protocol
7. Dilute the Fibronectin in sterile Hank’s Balanced Salt Solution (HBSS). 

Add 2 µL to 998 µL 

SECTION: LUHMES coating protocol
9. Rinse 3 times with HBSS.
SECTION: LUHMES coating protocol
10. Air dry for 15 minutes and add fresh LUHMES growth media. 
SECTION: LUHMES coating protocol
2. Dilute Poly-L-Ornithine solution to 50ug/mL in Nuclease-free water

Add 500 µL of PLO for every 500 µL Nuclease-free water

SECTION: LUHMES coating protocol
3. Add 7mL of the 50ug/mL PLO to a T-75 overnight at RT.
SECTION: LUHMES coating protocol
4. Rinse flask 3 times with  Nuclease-free water.
SECTION: LUHMES coating protocol
5. Allow the flask to air dry for 15 minutes uncapped and standing upright in the hood. (turn on UV)
SECTION: Cryopreservation
20. Label 2mL cryovials with the date, name, FIV#, Qbench number, passage number and cell type.     
 
Add about 1.5 million cells per vial with 1mL freezing media. 

Freezing Media: 
 7mL LUHMES Media
 4uL B-fgf (40ng)
 2mL FBS (20%)
1mL DMSO (1%)
SECTION: LUHMES Passaging (~ every 2-3 days)
14. Add fresh culture media to newly coated flask for at least 15 minutes before seeding cells to
allow media to reach normal pH
SECTION: LUHMES coating protocol
8. Place fibronectin coated flask in incubator for 3 hours
SECTION: LUHMES Differentiation
21. Day 0, when the LUHMES are 80% confluent, add differentiation media and incubate overnight.
SECTION: LUHMES Differentiation
22. Day 1, replate cells onto a new poly-l-ornithine and fibronectin-coated plate. Replate cells at a density of 1 million cells per T-75 flask or ~ 400,000 per well of a 6-well.
SECTION: LUHMES Differentiation
23. Replace LUHMES differentiation media every day while differentiating. LUHMES become mature dopamine-like neuron in 7-days.

 
**See material section for media concentration information**