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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>
<br>
Good luck,<br>
Vladan<br>
<br>
<div class="moz-cite-prefix">On 19/01/2022 16:50, Daniel Serwas
wrote:<br>
</div>
<blockquote type="cite"
cite="mid:33F3DEF6-3339-4540-878A-54598BFEE1A9@berkeley.edu">
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8">
Dear Leanne,
<div class=""><br class="">
</div>
<div class="">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.</div>
<div class=""><br class="">
</div>
<div class="">All the best,</div>
<div class="">Daniel</div>
<div class=""><br class="">
</div>
<div class=""><span style="orphans: 2; text-align: -webkit-auto;
widows: 2; -webkit-text-decorations-in-effect: none;" class="">Daniel
Serwas, PhD </span></div>
<div class="">
<div style="color: rgb(0, 0, 0); letter-spacing: normal;
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after-white-space;" class="">
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class="Apple-style-span" style="border-collapse:
separate; font-variant-ligatures: normal;
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normal; font-variant-alternates: normal;
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<div style="word-wrap: break-word; -webkit-nbsp-mode:
space; -webkit-line-break: after-white-space;"
class="">Postdoctoral Fellow</div>
<div style="word-wrap: break-word; -webkit-nbsp-mode:
space; -webkit-line-break: after-white-space;"
class="">Drubin Lab<br class="">
Department of Molecular and Cell Biology<br class="">
University of California, Berkeley<br class="">
610 Barker Hall<br class="">
Berkeley, CA 94720<br class="">
<br class="">
<br class="">
</div>
</span></div>
</div>
</div>
</div>
</div>
<div>
<blockquote type="cite" class="">
<div class="">On Jan 19, 2022, at 7:13 AM, Jager, L.A.H. de
(Leanne) <<a href="mailto:l.a.h.dejager@uu.nl" class=""
moz-do-not-send="true">l.a.h.dejager@uu.nl</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div class="WordSection1" style="page: WordSection1;
caret-color: rgb(0, 0, 0); font-family: Helvetica;
font-size: 12px; font-style: normal; font-variant-caps:
normal; font-weight: normal; letter-spacing: normal;
text-align: start; text-indent: 0px; text-transform: none;
white-space: normal; word-spacing: 0px;
-webkit-text-stroke-width: 0px; text-decoration: none;">
<p class="MsoNormal" style="margin: 0cm 0cm 8pt;
line-height: 15.546667098999023px; font-size: 11pt;
font-family: Calibri, sans-serif; text-align: justify;"><span
class="" lang="en-NL">Dear all,<span
class="Apple-converted-space"> </span><o:p class=""></o:p></span></p>
<p class="MsoNormal" style="margin: 0cm 0cm 8pt;
line-height: 15.546667098999023px; font-size: 11pt;
font-family: Calibri, sans-serif; text-align: justify;"><span
class="" lang="en-NL">In our lab we have grown
mammalian cells successfully on holey carbon R2/2 200
mesh gold</span><span class="" lang="en-NL"><span
class="Apple-converted-space"> </span></span><span
class="" lang="en-NL">grids</span><span class=""
lang="en-NL"><span class="Apple-converted-space"> </span></span><span
class="" lang="en-NL">(Quantifoil)</span><span
class="" lang="en-NL"><span
class="Apple-converted-space"> </span></span><span
class="" lang="en-NL"> 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.<o:p class=""></o:p></span></p>
<p class="MsoNormal" style="margin: 0cm 0cm 8pt;
line-height: 15.546667098999023px; font-size: 11pt;
font-family: Calibri, sans-serif; text-align: justify;"><span
class="" lang="en-NL">To figure out what was going on,
we conducted an experiment where we in parallel seeded
cells on glass, continuous carbon (gold, 200 mesh</span><span
class="" lang="en-NL">, Quantifoil</span><span
class="" lang="en-NL">) or the presumably faulty</span><span
class="" lang="en-NL">/toxic</span><span class=""
lang="en-NL"><span class="Apple-converted-space"> </span>R2/2
(gold, 200 mesh</span><span class="" lang="en-NL">,
Quantifoil</span><span class="" lang="en-NL">) 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 for</span><span class="" lang="en-NL">the</span><span
class="" lang="en-NL"><span
class="Apple-converted-space"> </span>washing
protocols looked smeared out/bloated. <o:p class=""></o:p></span></p>
<p class="MsoNormal" style="margin: 0cm 0cm 8pt;
line-height: 15.546667098999023px; font-size: 11pt;
font-family: Calibri, sans-serif; text-align: justify;">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 class=""
lang="en-NL">Alternatively, would it be useful to try
on Protochips c-flat grids instead?</span><span
class="" lang="en-NL"></span><o:p class=""></o:p></p>
<p class="MsoNormal" style="margin: 0cm 0cm 8pt;
line-height: 15.546667098999023px; font-size: 11pt;
font-family: Calibri, sans-serif; text-align: justify;"><span
class="" lang="en-NL">Kind regards,<span
class="Apple-converted-space"> </span><o:p class=""></o:p></span></p>
<p class="MsoNormal" style="margin: 0cm 0cm 8pt;
line-height: 15.546667098999023px; font-size: 11pt;
font-family: Calibri, sans-serif; text-align: justify;"><span
class="" lang="en-NL">Leanne</span><o:p class=""></o:p></p>
<p class="MsoNormal" style="margin: 0cm 0cm 8pt;
line-height: 15.546667098999023px; font-size: 11pt;
font-family: Calibri, sans-serif;"><o:p class=""> </o:p></p>
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