While I've not had that specific experience, I have done astrocyte-conditioned medium with brain capillary endothelial cell monolayers. There are a minimum of two experiments that need to be done. One is the percentage of conditioned medium and this depends upon the recipient cell and what is tolerated. We tested a range of dilutions. The other is the time span for conditioning. Again, we tested a range of times that obviously can effect the optimal dilution for the end point measured. Thus, plating density and growth conditions should be rigorously kept. Good luck and happy experimenting!

As Thomas mentioned, you will want to do some preliminary testing to find the optimal treatment paradigm for your studies depending on how long you want them in culture.

I have experience with ACM onto neurons, but each experimental design is different. I would recommend looking into using the Neurobasal media for the ACM so that you can directly add them to neurons, similar to what is used for astrocyte-neuron co-cultures.

As for the serum question, I will point you towards Ben Barres paper for the culturing of astrocytes: Development of a method for the purification and culture of rodent astrocytes. Neuron, 2011.

But my recommendation is to use pure Neurobasal media for the ACM so you can directly add to the neuronal cultures. Hope this helps!

I believe, it also depends on the sources of astrocytes; hiPSCs-derived astrocytes, fetal-derived astrocytes, mouse astrocytes and rat astrocytes they all behave differently with different media conditions along with serum conditions as well . Please refer to these three articles: http://jgp.rupress.org/content/jgp/149/1/149.full.pdf;

https://www.alzforum.org/news/research-news/purification-adult-human-astrocytes-shows-they-are-unique;

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