[3dem] mammalian cell freezing question for continuous carbon grids

[Anderson, Erik David]

Dear cryo colleagues,

I am wondering if anyone on this list has successfully frozen/imaged mammalian cells using continuous carbon grids, and if so whether they would be willing to provide tips regarding their freezing conditions?

Brief background, I’ve been trying to grow/image neurons (tried Hela as well) for tomography using continuous carbon grids of varying thickness (tested 3-10nm carbon layer, 150-200 mesh types, all gold), but have not had success during freezing step. I’m using a Leica EMGP, and have attempted back and front blots, respectively (tested 5-20 sec blot times, as well as different aliquots of media from 0-2uL, in 90-99% humidity). I’ve even tried side blotting, but still no success. All attempts have resulted in either broken grid holes or ice that is too thick (back blot generally causes more breakage, while front generally causes ice that’s too thick). I’ve verified the carbon remains (mostly) intact prior to freezing (at least much more intact than what is seen after freezing), and am mindful of how I take the grid out of the liquid prior to freezing. I should also add I’ve successfully grown/imaged the neurons on holey carbon grids, which are my (and it seems the literature’s) preferred grids for this system, but our order is delayed, and so that’s why I’m trying to make continuous carbon work.

If anyone has had success with these grids to image any kind of mammalian cell I’d be very appreciative of any advice/tips!

Thank you,

Erik Anderson
MD-PhD Candidate | Baylor College of Medicine
B.S. Microbiology and Music | University of Wisconsin-Madison | 2015
Ludtke Lab<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.bcm.edu_research_faculty-2Dlabs_steven-2Dludtke-2Dlab&d=DwIF-g&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=0pToNIycF_Lo1X5zhTGdAjwpMQnFME8apFLnrmQ7x6eIuOOouehtrBpEXZZVf5FJ&s=fil6vpim5X2wVq7qP-h05TIMTBY6OX5uLrldcv_CcKg&e= >


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