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Differentiation


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#1 tulip

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Posted 26 March 2013 - 03:35 PM

Does anybody have experience with differentiating smooth muscle cells like A7r5?

#2 jerryshelly1

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Posted 26 March 2013 - 06:37 PM

http://www.atcc.org/

Go to the above site and find your cell type. They will provide all the relevant information and additional published data outlining how your cells were used.

Diff of muscle cells is easy. Let cells go until they are confluent, change the rich media (10-15%), serum deprive them (1-5%) and let them differentiation for the desired number of days. Remember to change the media regularly as muscle can contract (as myotubes are forming) and pull off of the plate. If this happens, collagen or gelatin can fix this issue (for extensive differentiation, weeks).

#3 tulip

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Posted 26 March 2013 - 09:11 PM

Thanks for the info. I seeded the cells on matrigel coated coverslips and waited till they are confluent (1 week) and then I changed the medium to 1% serum. I waited almost 3 weeks after I switched to low serum and the cells were coming off from the coverslip, mostly they were rounded. so do you think collagen or gelatin would be a better substrate?

#4 jerryshelly1

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Posted 27 March 2013 - 07:26 AM

Matrigel coated cover slips are very analogous to collagen. I have never used them, but in the literature it appears as though they are the same or superior to collagen coating.

If your cells are lifting off of the plate, there are probably two things occurring. 1) You are not replacing the media frequently enough. When cells are switched from proliferation, to differentiation the amount of media consumed is more rapid. I would review the literature, or contact an author on the ATCC website to see if they noticed this problem. You could probably eleviate this by changing the media more frequently or placing your coated coverslips in a larger volume tissue culture dish. 2) It may seem trivial, but the method you handle your cells has a large impact. When you do change the media, are your extremely gentle with your pippetting? How long are you leaving your cells outside of the incubator? Is your media at the correct temperature when it is added to your differentiating cells? Have there been any fluctuations in your incubators CO2, O2, temperature? Just be mindful of these issues in the future and you should be fine.

Good luck




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