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

[Daniel Serwas]

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> 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
> 
>  
> 
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