I have been trying to produce an assay for the outgrowth of PC12's with NGF. I'm struggling to get a monolayer of cells in a 96 well assay plate coated with poly D lysine and I've also tried a PLL and laminin combination ( I absolutely batter them with a pipette, titrating them but this hasn't helped). Any suggestions would be greatly appreciated!!
It was suggested that I grow the cells adherently on Poly D lysine (from a cell biologist at ECACC) but I found even the dead cells stick to this. Can anyone suggest a better method?
Hi Rose,
With regards to coating wells, you may find the items from Sigma helpful. The growth surface area of each well in a 96-well plate is approximately 0.32 sq.cm, following manufacturer's instructions, you can adjust the volume accordingly. I also air-dry the wells I coated with collagen.
122-20 SIGMA | Rat Tail Collagen Coating Solution (20 ML)
125-50 SIGMA | Collagen Coating Solution (50 ML)
Sincerely,
John
http://www.sigmaaldrich.com/catalog/product/sigma/12220?lang=en®ion=PH
http://www.sigmaaldrich.com/catalog/product/sigma/12550?lang=en®ion=PH
Dear Rose,
I agree with Kwok-Hung. I have always grown the PC12 cells on collagen. We used acid-solubilized rat tail collagen that was just air-drid onto the plates, but we were working in a larger plate format. I hope someone here on RG can give you some tips for coating those tiny wells with collagen.
Good Luck!
Jill
Hi Rose,
With regards to coating wells, you may find the items from Sigma helpful. The growth surface area of each well in a 96-well plate is approximately 0.32 sq.cm, following manufacturer's instructions, you can adjust the volume accordingly. I also air-dry the wells I coated with collagen.
122-20 SIGMA | Rat Tail Collagen Coating Solution (20 ML)
125-50 SIGMA | Collagen Coating Solution (50 ML)
Sincerely,
John
http://www.sigmaaldrich.com/catalog/product/sigma/12220?lang=en®ion=PH
http://www.sigmaaldrich.com/catalog/product/sigma/12550?lang=en®ion=PH
Dear Rose
Attach PC12 cells on POLY-L-LYSINE or PLL(0.01%) solution. Coat fresh PLL on flask or plates with approx 10-15 minutes and wash plates or flasks two-three times with autoclaved water and store at 37.C . Use coated plates within one week.
Hope it works....
All the best !!!
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