Hi all,
I am going to try 3D culture (on top) with MCF10A cells. I am planning to start with matrigel and regular cell culture dishes and then perform confocal microscopy. Can anyone help me with the following points-
1. What should be the cell density of stable MCF10A cells (transfected and drug-selected) to seed on top of matrigel (according to dish size)?
2. After seeding how can I exactly observe acini formation? Do I need a specifically featured microscope? Or regular microscopes are fine?
3. If acini are formed (I hope positively), how acini are to be detached from matrigel? should I grow the cells on coverslips? Or how am I supposed to transfer the acini onto the slides?
Thanks in advance,
Swarnali
Hi Swarnali,
I can only answer your 1st question.
Please refer to this link (though not specific to 3D cultures, it is generally helpful for cell cultures): https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
Hope this helps.
All the best.
Hi Swarnali,
as to 2, you should be able to visualize acini formation through a phase contrast microscope. I used to visualized endothelial tube formation that way. For 3, you would only need to set up cells on slides/coverslips if you need to do downstream analysis like immunostaining. This can be done by purchasing Lab Tek chamber slides (1-8 chambers), an expensive option, or by sterilizing coverslips and placing them them in 6-well dishes for coating with Matrigel. Regardless, cultures on Matrigel are fragile so I’d look up specific protocols for fixation. Good luck!
Thank you Susan C Hubchak for your useful reply. I'll definitely try like you said.
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