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[jlolsen]

Hello,
  I am trying to differentiate human iPS cells into dopamine neurons.  I am following the protocol from Nature published by Kriks et al.  I am able to plate my NPCs onto polyornithine/laminin/fibronectin coated plates and coverslips and I observed good attachment of my cells.   Approximately 1 week after attachment, my cells become very loose and detach from the plate.  The last time this happened, I seeded the detached cells onto a newly coated plate and observed good attachment.  But after a week, these cells also detached.  I am beginning to suspect that my coating is being washed off my plates and coverslips each time I change my media.  Has anyone else ever experienced this problem?  Please see my coating protocol below:

Poly-L-ornithine (Sigma P4957)
Laminin (Sigma L2020)
Fibronectin (Sigma F1141)
3 6-well plates, 9 coverslips
20ug/ml Polyornithine: Combine 11ml of 0.01% polyornithine + 44ml PBS.
1. Add 2ml per well of 6-well plate and 1ml to each coverslip  
2. Incubate overnight at room temperature.
Next Day
1ug/ml laminin & 2ug/ml fibronectin:  55ul of 1mg/ml laminin +  110ul of 1mg/ml fibronectin + 55ml PBS
1.  Aspirate polyornithine coating and wash 3X with PBS
2.  Add laminin/fibronectin (2ml per well, 1ml per coverslip)
3.  Incubate 1 hr at 37C
4.  After the hour incubation, store at 4C wrapped in parafilm (do not aspirate until ready to use, no washing necessary, do not allow to dry out)

Any suggestions or input would be greatly appreciated!