I cross-link coverslips with gelatin, glutaraldehyde and ethanolamine and then I seed them with NIH-3T3 cells. They grow fine but when I lyse them they remain on the surface of coverslip-Why? How can I avoid that? I use 0.5% triton X and 20 mM Nh4OH in PBS as lysis buffer.
Krishna kumar Ganta
When you coat the coverslips with gelatin do you wash the excess gelatin ? . I suspect the high concentration of gelatin. Try using poly lysine or low concentrations of gelatin or the same concentrations with out gkutaraldehyde. Glutaraldehyde fixes amines to the surface. Ideally u need to wash excess gelatin and ethanolamine followed by a fix and washes with pbs to remove excess glutaraldehyde. Let us know ur coating protocol and ur purpose of seeding on coverslips ??
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