Polyornithine coating

1. In a sterile hood, pour out contents of 50 mL tube containing sterilized coverslips into the lid of a six-well plate. (This lid works well as a basin to pick up coverslips.)

2. Using sterilized forceps, pick up one coverslip at a time and set upright in a well of a 24-well plate. Repeat for the whole plate. Be careful that there are not two coverslips stuck together and try not to scratch the coverslips.

3. Allow coverslips to dry completely.

4. Tap plates until coverslips have fallen flat in each well.

5. Add 100 pL polyornithine (0.1 mg/mL in sterile dH2O) to each coverslip. Keep the drop on the coverslip alone.

6. Incubate plates at 37°C overnight.

7. Remove plates from the incubator and allow to cool to room temperature before proceeding.

8. Aspirate polyornithine off of each coverslip. Do not scratch/touch the center of the coverslip. Touch only the edge of the coverslip when aspirating.

9. Allow coverslips to dry for approximately 30min before washing.

10. Add 1 mL sterile dH2O to each well.

11. Let sit for 10min.

12. Aspirate water from each well.

13. Repeat steps 10 and 11 two more times, for a total of three washings.

14. Allow plates to completely dry by removing the lids and leaving them in the hood as long as necessary.

15. Cover plates, wrap in foil, label with date and store at —20°C. Alternatives to laminin-enhanced adhesion

Attachment of ESC aggregates or neuroepithelial clusters can be accomplished with a variety of different adhesion molecules including fibronectin and Matrigel™. Another quick and cost-effective method to facilitate adhesion is to supplement neural induction medium with 10% fetal bovine serum (FBS) for 12-24 h. The serum should then be washed away after the aggregates have attached. Although serum should be avoided for neuroepithelial differentiation, this short exposure to enhance adhesion does not significantly reduce the overall efficiency of the culture system for generating neuroepithelial cells. It should be noted that the use of serum may affect some gene expression patterns.

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  • giordano
    How to weigh polyornithine?
    8 years ago

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