The astrocytes I have in culture are growing too slowly for experimentation - is there anything that can be done to make them divide faster?
Hi Indrani,
Are you working with primary astrocytes or an astrocyte cell line?
Sincerely,
Monica Holley
Hello Dr. Holley and Mr. Schmitt,
Thanks for the help! I am working with the astrocyte cell line C8-D1A. My experiment involves transfecting the cell line with a microRNA (which I have done), and then isolating exosomes from the cells. I am a bit concerned that I may not be able to isolate enough exosomes; also, I'm having difficulty in producing a confluent plate (in 24 wells) for a scratch assay.
I am currently using DMEM w. 10% serum, GlutaMAX, and penstrep. I have also ordered an N-2 Supplement, found here: https://www.lifetechnologies.com/order/catalog/product/17502048
Is there any chance this supplement will promote growth?
Thank you!!
Indrani
Hello Dr. Hancock,
Thank you for the advice! I believe my DMEM is high glucose - and 10% serum with penstrep. And, I am incubating them at 5% CO2.
I have had this culture for about two weeks now, and they seem to be ready for experimenting, although I still have difficulty reaching confluency. I have attempted a stable transfection with miRNA; hopefully, they will have been replicating enough for that to work. Either way, I'll know once I use selecting media...
Thank you!!
Indrani
I also grow C8-D1A ( astrocyte type I cone) . I have also had bad experiences working with these cells. I found they are not a stable cell line!!! I think ,this cell line is very sensitive to trypsin. There are plenty of times, they are killed by trypsin. I now start getting rid of trypsin by spinning every time I subculture them. So think about it if you are using trypsin while subculturing.
I recommend you to use primary cultures. I have had great experiences with micro RNA transfection in primary astrocytic cultures.
For mouse: Neural Tissue Dissociation Kit-P and ACSA-2 MicroBeads
For rat: Neural Tissue Dissociation Kit-T and ACSA-1 (GLAST) MicroBeads
Acute isolation prevents modifications of the cells that occur during the shake-off method.
Thank you once again, to Mr. Kuwar, Mr. Diniz, and Dr. Pennartz!! I really appreciate all the great advice, and as time goes on, I will definitely incorporate them.
Thanks!
Indrani
If you are interested, these my two papers present the protocol of purified astrocytes cultures.
http://www.ncbi.nlm.nih.gov/pubmed/23055518
http://onlinelibrary.wiley.com/doi/10.1002/glia.22713/abstract
Mycoplasma-contaminated cells have a slow growth rate. I suggest you do a test on their cells.
kind regards,
Luan.
i woild like to share my astrocytes culture protocol here for your convenience
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