Can you explain your experiment a little bit better? Are these U87 tumorspheres? Are those 3d-cultures on chamberslides?
Thank you for entering my dilemma. The actual 3D plates you can found at http://www.reinnervate.com. They state that you can grow your cultures in 3D (in a matrix) and then you can investigate them in confocal microscopy after labeling whatever. The question was (from your experience) if the auto-fluorescence of the matrix is high? How do you actually handle the matrix to put it on the slide, and so on. We are starting to use these 3D plates for U87 that make on normal 2D cultivation microspheres.
I would try low adhesion plates with their normal culture medium to avoid the matrix. I tried it with U251, they form nice spheres in the low adhesion plates. Its also cheaper than the Matrix/the scaffold. At least what they stated on their website its a 3d-polystyrene scaffold. Its very hard to do imaging on a scaffold. In addition cancer cells having a high autofluorescence in the GFP (FITC) channel, so if you want to do 3d analysis avoid staining with FITC or Alexa 488. For the low adhesion stuff: Just use certain maybe 10.000 cells ? per well of a 48 well low adhesion plate and place them in the incubator over night, they should form spheres. With U251 it works fine. You can also try a softagar aproach, there should be a lot of literature out there.
Hope that helps.
Barbara
Wonderful, I will try the low adhesion plates for my U87 and (without any high hopes) just very few scaffolds as they are expensive and, if your indicate, probably the imaging in matrix is very difficult.
Thank you so much, it is always good to talk to professionals,
Kind regards,
Monica
Hi Monica, please keep us updated on the success of your approach, I may be interested in trying this also!
Thank you Barbara! As soon as I have some new information on this matter I will keep you up-dated with goods and bads overall.
Monica
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