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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Dear Leanne,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">You could try flame sterilization of grids like here:
<a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jove.com_t_50783_preparation-2Dprimary-2Dneurons-2Dfor-2Dvisualizing-2Dneurites-2Dfrozen-2Dhydrated&d=DwMGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=hjk4RHz3eJTc3jvr2f3_Pde2nonGX2aQbQEueBZLt5grK2qonbMi4eo2Pze4m1xq&s=pXx-nPsZE6iIiD_mqOy6xQ6ArgpcqrxrQV0cWaImw6A&e=">
https://www.jove.com/t/50783/preparation-primary-neurons-for-visualizing-neurites-frozen-hydrated</a><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">I used that for primary neurons, no coating or anything else needed, just sterilize shortly before seeding cells.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Best,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Julika<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif">From:</span></b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"> 3dem <3dem-bounces@ncmir.ucsd.edu>
<b>On Behalf Of </b>Vladan Lucic<br>
<b>Sent:</b> 19 January 2022 22:09<br>
<b>To:</b> Daniel Serwas <daniel.serwas@berkeley.edu>; Jager, L.A.H. de (Leanne) <l.a.h.dejager@uu.nl><br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Trouble seeding cells on R2/2 gold grids<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt">Dear Leanne,<br>
<br>
We had a similar problem, more than ten years ago, from one batch of grids to another our cells (neurons) did not grow well on quantifoil grids anymore. Turned out the reason was the manufacturer changed a washing step or something, but it took almost a year
to get good grids again. <br>
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Good luck,<br>
Vladan<o:p></o:p></p>
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<p class="MsoNormal">On 19/01/2022 16:50, Daniel Serwas wrote:<o:p></o:p></p>
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<p class="MsoNormal">Dear Leanne, <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">I normally wash grids with acetone and ethanol and incubate them in cell culture media over night in a cell culture incubator. At the next day, I replace the media with fresh one and seed the cells. For some cell types, I coat the grids
for example with poly-lysine or gelatin. How long have your cells been in culture? I made a similar experience that cells wouldn’t spread well on holey carbon anymore while still looking fine on glass or plastic when they reach a certain passage number.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">All the best,<o:p></o:p></p>
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<p class="MsoNormal">Daniel<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Daniel Serwas, PhD <o:p></o:p></p>
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<p class="MsoNormal"><span style="color:black">Postdoctoral Fellow<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span style="color:black">Drubin Lab<br>
Department of Molecular and Cell Biology<br>
University of California, Berkeley<br>
610 Barker Hall<br>
Berkeley, CA 94720<br>
<br>
<o:p></o:p></span></p>
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<p class="MsoNormal">On Jan 19, 2022, at 7:13 AM, Jager, L.A.H. de (Leanne) <<a href="mailto:l.a.h.dejager@uu.nl">l.a.h.dejager@uu.nl</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Dear all,<span class="apple-converted-space"> </span></span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;text-align:justify;line-height:11.65pt">
<span style="font-size:11.0pt;font-family:"Calibri",sans-serif">In our lab we have grown mammalian cells successfully on holey carbon R2/2 200 mesh gold<span class="apple-converted-space"> </span>grids<span class="apple-converted-space"> </span>(Quantifoil)<span class="apple-converted-space"> </span> for
some time now. However, a few months ago we started having problems with cells not attaching, spreading, or not looking happy on the grid. In case the mitochondria of the cells were stained with MitoTracker, these sometimes looked normal, but often smeared
out/bloated. This differed greatly per experiment. We hypothesized that something might be wrong with the grids.</span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;text-align:justify;line-height:11.65pt">
<span style="font-size:11.0pt;font-family:"Calibri",sans-serif">To figure out what was going on, we conducted an experiment where we in parallel seeded cells on glass, continuous carbon (gold, 200 mesh, Quantifoil) or the presumably faulty/toxic<span class="apple-converted-space"> </span>R2/2
(gold, 200 mesh, Quantifoil) and stained with MitoTracker. Some grids were washed either in ethyl acetate/ethanol/MilliQ or ethyl acetate/acetone/chloroform and ethanol/MilliQ prior to seeding. The mitochondria of the cells seeded on glass and on the continuous
carbon looked fine, but the mitochondria of the cells on the R2/2 both unwashed and forthe<span class="apple-converted-space"> </span>washing protocols looked smeared out/bloated. </span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;text-align:justify;line-height:11.65pt">
<span style="font-size:11.0pt;font-family:"Calibri",sans-serif">We were wondering whether anyone else lately has had trouble with cell seeding on grids or has any suggestions on other washing protocols?<span class="apple-converted-space"> </span></span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Alternatively,
would it be useful to try on Protochips c-flat grids instead?</span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;text-align:justify;line-height:11.65pt">
<span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Kind regards,<span class="apple-converted-space"> </span></span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;text-align:justify;line-height:11.65pt">
<span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Leanne</span><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:11.65pt"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"> <o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">_______________________________________________<br>
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<o:p></o:p></p>
<pre>_______________________________________________<o:p></o:p></pre>
<pre>3dem mailing list<o:p></o:p></pre>
<pre><a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><o:p></o:p></pre>
<pre><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><o:p></o:p></pre>
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