Dear You!

I used anti-GLAST antibody and microbeads for magnetic separation, but the principle is similar. I obtained viable cells with high purity.

Best wishes, Klara

Try to stain with Aquaporin4 as that might be more specific than GFAP. We detect GFAP+ cells in adult neural stem cells as well as in oligodendroglial progenitor cells... 

The magnetic beads method that Klara suggested is the way to go, that is positive selection. I have used a negative selection approach, where I use CD11b beads to remove the microglia from the astrocytes (I discard the microglia), It has worked exceptionally well (less than 0.5% CD11b positive cells by FACS after negative selection). Below is a paper from a colleague in the lab that briefly describes the method that I have used for my own work.

Negative selection may be preferred in the instance where you want to avoid inadvertent receptor "activation" in cells  (astroglia in your case) that may be caused by antibody binding. this is the reason why we discard the microglial cell after binding to CD11b. We use a different method for purifying microglia from astroglia that does not involve an immuno-technique.

I would probably avoid your approach of using FACS to sort your cells as I have found issues with viability.

I can provide you with a protocol if you like. Let me know.

Best of luck.

The link did not appear. Here it is again.

http://www.ncbi.nlm.nih.gov/pubmed/17902170

@ Björn, yes, I now noticed that You Li specifically asked about cells from adult. Nevertheless, I hope she can glean something useful from my responses. I do not have experience in isolating from adult mice, but I assume a similar approach is possible. We considered isolating from adult (since we expect differences between neonatal glia and adult glia), but never got around to it. Perhaps a density gradient would be the best approach followed by immuno-selection, I imagine someone has tried this.

It would be interesting to see if anyone has attempted a similar approach with adult tissue, though the complicating factor is the highly vascularized nature of adult CNS and the mature meninges, which is difficult to remove from adult.

Best.

Björn, I had a look at the other thread. I had to chuckle a little, because I recall that many of the concerns and problems people shared in that thread are probably the same reasons why we decided not to pursue adult cells. We had plenty of work using the neonatal cells and we generally have no trouble publishing with those, especially if we can corroborate with some type of in vivo evidence (e.g. IHC or in situ hyb on brain sections).

If I was going to start a similar project now, besides the gradient I might try an explant culture and perform the purification from the cells that attach and migrate from the explant. I suppose you might get a fair number of astroglia this way and can avoid FACS as even from the neonatal isolation I noticed that we had an issue with viability. Seems to me that culturing the cells will involve something other than FACS. The magnetic beads method might be preferred, but as noted above, I prefer the negative selection and you may need to do positive selection to isolate from an explant culture.

Hope something works for you.

Isolating astrocytes from adults is tough because everything is so integrated.  You might try papain digestion and immuno-panning since this is more gentle than dounce homogenizing, gradient, and sorting.  When I use the homogenizer on adult brains it kills most of the cells.  Good Luck!

@ Ricardo and Klara:

thanks for your suggestions and thank Ricardo for your reference. Magnetic separation is a good way to try. My problem is that I am not sure whether the cd11b- cells are astrocyte using my dissociation method. I tried to gate cd11b- cells with anti-GLAST- PE. but they are GLAST-negative. Then i stained cd11b- cells with GFAP. I got both cd11b- and + stained with GFAP. So my next first thing is to confirm that adult astrocyte can be stained by anti-GLAST and the cd11b- cells dissociated by my protocol are astrocyte.

my protocol to dissociate the astrocyte:

mince the brain

digest with collagenase IV  and dispase and DNAseI for 1h

40% percoll to remove the myelin and collect the pellet.

stain with cd11b and cd45

Thanks for your help and glad to discuss with you.

@ Björn  Thanks for your answer. I think we are doing the same thing--adult astrocyte and microglia dissociation? I just tried, but did not get any anti-GLAST staining with CD11b- population. Here is my protocol to dissociate the cells:

mince the brain

digest with collagenase IV and dispase and DNAseI for 1h

40% percoll to remove the myelin and collect the pellet.

stain with cd11b and cd45

Could you have a look at it and is there any problem with this protocol? I will try the BSA gradient instead of percoll. 

I am going to do some study about the astrocyte- microglia communication to see if there is some change in our model. So FACS is fast way to check. 

Thanks,

Thank everyone so much for all of your answers. They are really helpful to me.