<html><head><meta http-equiv="Content-Type" content="text/html; charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; line-break: after-white-space;" class="">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; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div style="color: rgb(0, 0, 0); letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div style="color: rgb(0, 0, 0); letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div style="color: rgb(0, 0, 0); letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><span class="Apple-style-span" style="border-collapse: separate; font-variant-ligatures: normal; font-variant-position: normal; font-variant-numeric: normal; font-variant-alternates: normal; font-variant-east-asian: normal; line-height: normal; border-spacing: 0px; -webkit-text-decorations-in-effect: none;"><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="">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 lang="en-NL" class="">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 lang="en-NL" class="">In our lab we have grown mammalian cells successfully on holey carbon R2/2 200 mesh gold</span><span lang="en-NL" class=""><span class="Apple-converted-space"> </span></span><span lang="en-NL" class="">grids</span><span lang="en-NL" class=""><span class="Apple-converted-space"> </span></span><span lang="en-NL" class="">(Quantifoil)</span><span lang="en-NL" class=""><span class="Apple-converted-space"> </span></span><span lang="en-NL" class=""> 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 lang="en-NL" class="">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 lang="en-NL" class="">, Quantifoil</span><span lang="en-NL" class="">) or the presumably faulty</span><span lang="en-NL" class="">/toxic</span><span lang="en-NL" class=""><span class="Apple-converted-space"> </span>R2/2 (gold, 200 mesh</span><span lang="en-NL" class="">, Quantifoil</span><span lang="en-NL" class="">) 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 lang="en-NL" class="">the</span><span lang="en-NL" class=""><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 lang="en-NL" class="">Alternatively, would it be useful to try on Protochips c-flat grids instead?</span><span lang="en-NL" class=""></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 lang="en-NL" class="">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 lang="en-NL" class="">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></div><span style="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; float: none; display: inline !important;" class="">_______________________________________________</span><br style="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;" class=""><span style="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; float: none; display: inline !important;" class="">3dem mailing list</span><br style="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;" class=""><a href="mailto:3dem@ncmir.ucsd.edu" style="font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px;" class="">3dem@ncmir.ucsd.edu</a><br style="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;" class=""><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" style="font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px;" class="">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a></div></blockquote></div><br class=""></body></html>