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the grids. To determine what was going on, we conducted an experi- ment where, in parallel, we 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. Te mitochondria of the cells seeded on glass and the continuous carbon looked fine, but the mitochondria of the cells on the R2/2 grids both unwashed and for the washing protocols looked smeared or bloated. We wonder if anyone else has had trouble with cell seed- ing on grids or has suggestions on other washing protocols? Alterna- tively, would it be useful to try Protochips c-flat grids instead? Kind regards, Leanne Jager l.a.h.dejager@uu.nl


I normally wash grids with acetone and ethanol and incubate


them in cell culture media overnight in a cell culture incubator. Te next day, I replace the media and seed the cells. For some cell types, I coat the grids with poly-lysine or gelatin. How long have your cells been in culture? I had a similar experience where cells would not spread well on holey carbon grids while looking fine on glass or plastic when they reached a certain passage number. Daniel Serwas daniel.serwas@berkeley.edu


We had a similar problem when changing from one batch of


grids to another where our cells (neurons) did not grow well on Quantifoil grids. Turned out the reason was the manufacturer changed a washing step, but it took almost a year to get good grids again. Vladan Lucic vladan@biochem.mpg.de


You could try flame sterilization of grids. I used this for


primary neurons and no coating was needed. Just sterilize shortly before seeding cells. Julika Radecke julika.radecke@diamond.ac.uk


Question about Human Epidermoid Cancer Cell (HEp-2) Morphology Microscopy Listserver We have a TEM sample of control HEp-2 cells. In thin sections,


the cells have areas of various sizes where it appears the cytoplasm has been “removed,” for lack of a better word. At low magnifica- tion our first thought was that the cells were showing vacuolization. However, these areas are not membrane bound. Within the areas, the faint material looks like the surrounding cytoplasm. Tese cells have several lysosomes in various stages, but otherwise the cells ap- pear healthy. I do not think it is stain, fixation, or other preparation artifacts. Some cells have many of these areas while others have very few. Tere is no evidence of bacterial or viral contamination. Noth- ing was done experimentally to these cells, as baseline morphology is needed before doing experiments. Is it possible to have digestion or degradation of the cytoplasm in the absence of any kind of mem- brane bound digestive vesicle or vacuole? All help is appreciated. Tom Bargar tbargar@unmc.edu


How many times were these cells passaged? Many moons ago


I worked with HEp-2 cells, and I remember that they get really ugly aſter repeated passages. I was using them in light microscopy, and they had very large accumulations of “stuff” in them. Te solution was to use a lower passage number. I know that doesn’t answer your question directly, but it may not be anything you did once they were in your hands, rather that the cells were “too old”. Lee Cohen-Gould lcgould@med.cornell.edu


66 www.microscopy-today.com • 2022 May


Hep-2G cells are frequently filled with non-membrane-bound


lipid droplets. Many examples are out there via Google images. Tomas Phillips phillipst@missouri.edu


What buffer was used? Phosphate buffers may cause uneven


extraction of cytoplasm. Glutaraldehyde generally is less extractive. Phil Oshel oshel1pe@cmich.edu


Te only thing I can think of is that the cells showing this


morphological feature are dying. In this case, one can see strange intracellular structures which relate to nothing you expect to see. If this happens in only a few cells, it is possible to miss them in the light microscope. However, if most cells look like that (most cells are dying), this should also be recognizable under a light micro- scope to any person acquainted with these cells. I would ask these questions: How old is the cell culture? How many passages? Are the cells mycoplasm free? Another reason might be extraction during dehydration. In this case I would consider contamination in the dehydration solutions. Tis is easy to verify: prepare other, well- known cells in your laboratory with the same method and the same products and if they show the same “artifacts”, you have found the suspect! Stephane Nizets nizets2@yahoo.com


Do these cells have glycogen granules? Did you process them


with uranyl acetate en bloc? Uranyl acetate partially removes glyco- gen and creates empty areas in the cytoplasm. Hilda Amalia Pasolli amaliapasolli@gmail.com


Someone previously mentioned lipids. If OsO4 was not used,


then lipids would be extracted in subsequent solvents and liquid plastic. Jan Factor jan.factor@purchase.edu


Crossword Puzzle Answers See puzzle on page 58.


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