We had cultured, fixed, and stained cells directly in the wells of a standard 6-well tissue culture plate. However, we are realizing we need to do higher magnification to see some of the staining features, and because our confocal is inverted it would be best to punch out the wells or cut them out in some way so they can be mounted under a coverslip and imaged. Does anyone have a suggestion for ways to do this?
I did something comparable and used a fretsaw for this. Put some drops of PBS/glycerine in the wells first to avoid drying out of the cells. After sawing the bottom out, rinse it a few times with PBS and mount to a glass slide.
Good Luck
A better and easier solution to cutting out the bottom would be to culture the cells on glass coverslips and then take the coverslip out and mount it to do confocal. Fisher has a 25mm coverslip (Catalog #12-545-86) that is slightly smaller than the diameter of a well in a 6-well dish. You will have to use a non-tissue culture treated 6-well dish such that the cells adhere to the coverslip and not the well. We do this frequently in order to image cells by confocal and it works very well.
Good Luck!
Thanks! (Tom, will be doing this in the future of course...just did not plan for it this particular time) We have also been told that using a hot needle to cut through the plastic tends to work although is labor intensive. will report back.
Hi Justing:
No sure if you are aware or not, but they are commercially available (albeit overpriced!) https://www.mattek.com/store-category/cultureware/glass-bottom-multi-well-plates/.
A cheap alternative we have done in the past, is to simply heat up a cork borer (i.e. the metal tool for cutting a hole in a cork or rubber stopper) of the desired diameter and simply punch/melt a hole through the bottom of plate/well. Don't get the metal too hot or you will end up melting away a larger hole; with the right temperature you will get enough heat into the borer to help it move through the plastic smoothly.
Then just glue a round coverslip to the bottom (outside face) of the plate using sylgard 184 or your favorite adhesive. Works great for plating and imaging or as a recording chamber for patch clamp experiments.
Hope this helps. Good luck
Hi Justin,
I had this issue when I wanted to image my cells at high-mag using confocal (FV-3000 Olympus). Apparently, the bottom of multi-well plates are too thick for 20X and higher lenses. You can try growing your cells on #1 coverslips coated with poly-D-lysine (for cell attachment), and use them for imaging. coverslips can be mechanically supported with glass slide, and small amount of nail polish to fix in place.
I also tried cutting the bottom of either 6 or 24-w plates out with heated razor blade for SEM, but the heat might dry out your cells on the edges. This method is definitely more difficult on 96-well plates.
Best of luck
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