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I'm developing a cell-based assay in 96-well plates that requires adherent cells, as they need to be washed at least twice during the protocol. I'm using in-house strains of HT1080 cells (some overexpressing a certain protein of interest) that unfortunately have been selected for suspension growth in chemically-defined media (50/50 CD-CHO/CD-293). I can convince them to adhere by culturing in DMEM with 10% FBS and growing them in CELLBind flasks, but they still tend to pull away if there is very much shearing force, such as during the wash steps, or when they're in PBS during the assay wash and incubation steps (maybe 2 hours total for now, I'm still optimizing that).

Unfortunately, the parameters of my experiments don't allow me to keep the cells in serum-containing media during the assay, as it would interfere with the uptake of my drug. Like I've said, when in PBS they start detaching, and I would assume the same in HBSS, which I don't really want to use anyway due to its low buffering capacity in CO2 incubators.

Might DMEM alone (without FBS) provide a little more impetus to stay attached during the assay? What would the effect be of increasing the amount of FBS to say 15 or 20% during culturing - would that promote stronger attachment? I'm a little hesitant to coat the wells with poly-D-lysine, as my readout is on a fluorescent imager, and I'm worried it would substantially increase background. I don't really have enough time to re-select the cells for adherent growth (although if I'd known about these issues from my predecessor 3 or 4 months ago I would have started right away...), so are there any other tricks I can try in the meantime? Are there any media supplements I could use to promote adherin expression or something like that? Any tips or advice at all would be appreciated.

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I'd suggest splitting out your edit into a new question, it looks like it would be a fine question on it's own. –  Mad Scientist Jun 5 '13 at 6:29
    
@MadScientist - that's true, I hadn't thought of that. Coming right up! –  MattDMo Jun 5 '13 at 15:12
    
I don't remeber this anymore exactly, but weren't there cell culture plates available which had a membrane bottom which allowed you removing liquids by centrifugation while the cells stayed on the membrane? –  Chris May 5 at 15:26

2 Answers 2

My main concern here is why you are using suspension-culture selected cells for an adherent cell-based assay? Is it possible to obtain a clone of adherent HT1080? Depending on what you are assaying with your protein of interest, the cell biology may be altered changing from suspension/adherent culture systems.

Here are my experiences using PC12 cells in culture, which are weakly adherent cells, with a preference to grow in clumps, rather than monolayers. I have found that these cells have a preference for specific tissue plastic; (without mentioning names), these cells will adhere on some poly-D-lysine coated plates while they won't adhere on others. I routinely perform immunofluorescence microscopy with these cells, and grow them on glass cover slips which I have skim-coated with rat tail type I collagen. This coating provides minor background in the green range and only when imaging close to the glass. With a bright fluorophore (e.g., AlexaFluor or cyanin dyes for fixed cell; enhanced GFP for live cells), this backgorund is not a problem for quantitative microscopy.

Lastly, for secretion assays we use high-glucose DMEM supplemented with 1% dialyzed FBS. If memory serves, this retains all serum components less than ~15 kDa, which includes various growth factors. I have observed these cells continue to live for up to one day (and maybe longer?) in supplemented medium, while they die after a few hours without. This may also help with non-specific binding you may be worried about with drug addition.

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Thanks for sharing your experience. The main reason I'm using the cells is because that's what everyone else in the company is using, and they've been adapted to suspension growth for scaled-up production of ... stuff (pharma R&D). Some of the lines are various clones overexpressing a certain receptor, although after talking with my boss we might be able to work around that in our experimental design. My budget isn't that big, so I'm trying to work with what I've got, although if what we've got doesn't work we'll need to get something else :) –  MattDMo Jun 4 '13 at 22:52
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Just for the record, I hate PC12 cells. You're exactly right about them growing in some vessels, and not others, and only with very specific media and at very specific densities. They'll pull off the plate just to spite you, and if they're not dying every third passage they're getting contaminated. Good luck with them, I don't envy you! –  MattDMo Jun 4 '13 at 22:55
    
Thanks MattDMo, they took a good while to troubleshoot! The biggest challenge now is getting them at the right density for single cell imaging and not some gnarly unresolvable clump. ;) –  leonardo Jun 4 '13 at 23:09
    
try gelatin or collagen coating on the culture plates.. –  WYSIWYG Jun 10 '13 at 7:48
    
@WYSIWYG - I currently collagen coat the coverslips. The nature of PC12 cells is that they prefer to grow in close proximity to each other, and spontaneously form cell clumps as they grow. –  leonardo Jun 10 '13 at 16:01

Have you looked into trying the DropArray microplates? They're a new technology where you don't need to centrifuge your cells to wash them...an old colleague of mine works for the company and they're pretty cool about demoing the products/instruments for you. It's http://www.curiox.com/.

I think most people that use the system are using suspension cells- because of the challenges you listed above, so may be worth a look!

Good luck!

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Interesting. I'll have to check them out a little more. I'm not developing the assay anymore (I posted the question a year ago), but I ended up switching platforms to flow cytometry, and just used round-bottom/v-bottom plates. –  MattDMo May 6 at 3:05

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