[3dem] Carbon peeling from C flat gold grids

[Janet Meehl]

Hello,
I’m having issues with C flat grids that I recently purchased.  The grids are holey carbon 2/2 and 1.2/1.3 gold 200 mesh grids purchased from EM Sciences. Prior to seeding cells on grids, they are glow discharged, UV sterilized in a tissue culture hood, dipped briefly in 70% ethanol followed by water and placed in a culture dish. Poly lysine is added to the dish and grids sit overnight at 4C before being rinsed several times with water. Media and cells are added. Grids are plunge frozen using a Leica GP2 and are clipped to be imaged in a Krios microscope.  In the light microscope, carbon films appear intact prior to freezing but I think the carbon is very fragile and will tear upon the slightest perturbation.  In the last experiment, every grid (8) had large areas of carbon missing.  I have used Quantifoils for this same protocol with no issues at all.
Is anyone having problems with C flat grids in tissue culture conditions and if so, is there anything I should do to prevent carbon from peeling off the grid?
Regards,
Janet Meehl
Voeltz Lab/HHMI/University of Colorado

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