After a few days of cell cultur in 96 well ULA microplate, spheroids are easly obtained. The diameter of the spheroids measures approximately 200-300 µm. After that, medium cultur is carefully removed and spheroids are fixed with 4% PFA during 30 to 60 min at room temperature. My question is how to ensure that spheroids are centered and physically attached to the specific plastic of the ULA plate (to prevent them from moving during the staining protocol) ? I think it could be useful to cover fixed spheroids with a gel for rapid solidification (polyacrylamide, matrigel, collagen,...). Do you have advice about the gel to be used ? Thank you
After fixation, why not place in OCT and cryosection onto a coverslip? This worked well for me for staining of stem cell spheroid aggregates. I placed the the OCT in a 1.5 mL eppendorf tube cap and directly applied the spheroids into the cap. Start with a small layer of OCT, place the spheroids, and top off the cap with more OCT. Freeze this and cryosection onto a coverslip. Just an idea! Best of luck.
E
Thank you Elliot. It is a good idea ! Nevertheless, I want to stain spheroids directly in the ULA plate for automated cell identification by high content analysis.
Benjamin Kopp --- Gotcha... I've used matrigel in the past and it seemed to work fine with staining. Give it a go and let us know how it works.
Sounds like a fun project to work optimize. Best of luck!
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