20180514 – 20180522: HTB-11 cells diferentiation (exp. 3) + ICC

Day 0- 2018.05.14: Coating and seeding

• Prepare  plates of 60mm of diameter with 14 coverslips (12 mm)
• Treatment with Poly-D-Lysine (diluted 1/40 with PBS)
• Incubate for 1h (37ºC, 5% CO2).
• Wash with PBS 1x
• Treatment with Laminin (diluted 2 microL/ml PBS)
• Wash with PBS 1x
• Seed 4000 cells/cm2 –> every plate has 21 cm2 –> 84000 cells/plate 60 mm (in proliferation medium)
• Incubate at 37ºC for 24h.

*Proliferation medium:

• DMEM
• 10 % FBS
• 1 % Pen-Strep
• 1 % L-glutamine
• 1 % Sodium pyruvate

Day 1- 2018.05.15: treatment and medium exchange

Prepare differentiation medium and add retinoic acid.

• Differentiation medium:
  • Neurobasal –> 48 ml
  • 2 % B27 (stock at 50x) –> 1 ml
  • 1 % Glutamax –> 0’5 ml
  • 50 ng/ml NGF (stock at 100 µg/ml) –> 25 µl
  • 1 % Pen-Strep –> 0’5 ml
• Retinoic Acid (RA) 10 µM –> Stock solution -80ºC at  0,01 M –> 1 µl every ml of medium

Day 3- 2018.05.17: treatment and medium exchange

Same conditions as day 1

Day 4- 2018.05.18: treatment and medium exchange

Same conditions as day 1

Cells are well differentiated, but there are few of them –> Seed 10000 cells/cm2 instead of 4000 cells/cm2

Day 8- 2018.05.22: ICC (proof)

• Fix with PFA 4% (aprox 5 ml/plate). Incubation at RT during 10min.
• Wash with PBS 1x
• Blocking solution: Goat Serum 5 % in PBS for 1h at RT.
• Patient’s sera diluted 1/100 with 5 % Goat Serum.
• Secondary antibody: GAH488 IgG diluted 1/1000
• Vectashield mounting medium

Results: the staining in this neurons correlates well with the staining in rat DRG neurons,