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<div style="" dir="ltr">Hi all,</div>
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<div style="" dir="ltr">Thank you for the replies! @Chen I am using a Leica Emgp, but we do have a vitrobot, and so re Dr. Kirchweger’s suggestion will try using that. And thank you for the paper and sharing your experience Dr. Hu, I agree holey carbon seem
to work the best for this sort of system (I will likely just need to wait for our order to arrive). I had tried manual plunging when I did side blot, but perhaps I could try it with backblotting and see if that helps improve ice. </div>
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<div style="" dir="ltr">Thanks again for the replies and advice!</div>
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<div style="" dir="ltr">-Erik </div>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> Peter Kirchweger <peter.kirchweger@weizmann.ac.il><br>
<b>Sent:</b> Sunday, May 29, 2022 3:33:55 AM<br>
<b>To:</b> Anderson, Erik David <Erik.Anderson@bcm.edu>; 3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu><br>
<b>Subject:</b> Re: mammalian cell freezing question for continuous carbon grids</font>
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Dear Eric, <br>
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I can feel your pain ... I have also tried many things as you describe. <br>
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The only way I could at least partially successful plunge continuous carbon grids was by using the Vitrobot and by blotting from both sides.
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This, however, can create deroofed cells when doing that (which happened to me). Additionally, I usually only got half of the grid with good ice thickness, while the upper half was too thick.
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So, I stopped the continuous carbon again and went back to holey carbon.</div>
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Good luck and best regards ... <br>
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<span style="margin:0px; font-family:Arial,Helvetica,sans-serif; font-size:11pt">Dr. Peter Kirchweger</span></div>
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<div id="x_divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" color="#000000" style="font-size:11pt"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Anderson, Erik David <Erik.Anderson@bcm.edu><br>
<b>Sent:</b> Saturday, May 28, 2022 7:32 AM<br>
<b>To:</b> 3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu><br>
<b>Subject:</b> [3dem] mammalian cell freezing question for continuous carbon grids</font>
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<p class="x_x_MsoNormal"><span style="font-size:11.0pt">Dear cryo colleagues,</span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt"> </span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt">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?</span></p>
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<p class="x_x_MsoNormal"><span style="font-size:11.0pt">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.</span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt"> </span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt">If anyone has had success with these grids to image any kind of mammalian cell I’d be very appreciative of any advice/tips!</span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt"> </span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt">Thank you,</span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt"> </span></p>
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<p class="x_x_MsoNormal"><b><span style="font-size:11.0pt; font-family:"Times New Roman",serif; color:black">Erik Anderson</span></b></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt; font-family:"Times New Roman",serif; color:black">MD-PhD Candidate | Baylor College of Medicine</span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt; font-family:"Times New Roman",serif; color:black">B.S. Microbiology and Music | University of Wisconsin-Madison | 2015</span></p>
<p class="x_x_MsoNormal"><span style="font-size:11.0pt; font-family:"Times New Roman",serif; color:black"><a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__www.bcm.edu_research_faculty-2Dlabs_steven-2Dludtke-2Dlab&d=DwMF-g&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=0pToNIycF_Lo1X5zhTGdAjwpMQnFME8apFLnrmQ7x6eIuOOouehtrBpEXZZVf5FJ&s=fil6vpim5X2wVq7qP-h05TIMTBY6OX5uLrldcv_CcKg&e="><span style="color:#0563C1">Ludtke
Lab</span></a> </span></p>
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