[3dem] Trouble seeding cells on R2/2 gold grids

Wed Jan 26 08:27:14 PST 2022

Dear Leanne,

You could try flame sterilization of grids like here: https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jove.com_t_50783_preparation-2Dprimary-2Dneurons-2Dfor-2Dvisualizing-2Dneurites-2Dfrozen-2Dhydrated&d=DwIGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=hjk4RHz3eJTc3jvr2f3_Pde2nonGX2aQbQEueBZLt5grK2qonbMi4eo2Pze4m1xq&s=pXx-nPsZE6iIiD_mqOy6xQ6ArgpcqrxrQV0cWaImw6A&e= 
I used that for primary neurons, no coating or anything else needed, just sterilize shortly before seeding cells.

Best,
Julika

From: 3dem <3dem-bounces at ncmir.ucsd.edu> On Behalf Of Vladan Lucic
Sent: 19 January 2022 22:09
To: Daniel Serwas <daniel.serwas at berkeley.edu>; Jager, L.A.H. de (Leanne) <l.a.h.dejager at uu.nl>
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Trouble seeding cells on R2/2 gold grids

Dear Leanne,

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.

Good luck,
Vladan
On 19/01/2022 16:50, Daniel Serwas wrote:
Dear Leanne,

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.

All the best,
Daniel

Daniel Serwas, PhD
Postdoctoral Fellow
Drubin Lab
Department of Molecular and Cell Biology
University of California, Berkeley
610 Barker Hall
Berkeley, CA 94720

On Jan 19, 2022, at 7:13 AM, Jager, L.A.H. de (Leanne) <l.a.h.dejager at uu.nl<mailto:l.a.h.dejager at uu.nl>> wrote:

Dear all,
In our lab we have grown mammalian cells successfully on holey carbon R2/2 200 mesh gold grids (Quantifoil)  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.
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 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 washing protocols looked smeared out/bloated.
We were wondering whether anyone else lately has had trouble with cell seeding on grids or has any suggestions on other washing protocols? Alternatively, would it be useful to try on Protochips c-flat grids instead?
Kind regards,
Leanne

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

_______________________________________________

3dem mailing list

3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>

https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

-- 
This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail.
Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. 
Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message.
Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20220126/888ba27b/attachment.html>