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

[Vladan Lucic]

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 goldgrids(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/toxicR2/2 (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 forthewashing 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
>>
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