The expression of CD294 in peripheral blood mononuclear cells (PBMCs) is highly restricted to an activated state of Type 2 cells ; i.e. CD25 negative T cells do not express CD294 .

Cd 294 may be expressed weakly on some monocytes or dendritic cells !

nothing beats papain. I'd agree with the suggestion above. matrigel. Or try poly-ornithine/lamin or poly-lysine. depending on your despair...hehe...good luck.

Sorry, but its almost a guaranteed "no go" because like David said, astrocyte morphology (and function) really depend on there interactions in situ. He is also right that it doesn't matter if they are acutely isolated or cultured for long periods of time. In in vivo conditions, astrocytes look the way they do because each tiny process they have is wrapped around a synapse with the apical part of the astrocyte cell also protruding out to form part of the blood brain barrier. So, if you think about it, if you isolate astrocytes they will have neither neurons or vasculature to interact with to have a "normal" morphology. Also, if you do almost anything to an astrocyte or the area around an astrocyte you typical invoke a response called reactive astrocytosis or astrogliosis. A well documented hallmark of this response is the increased expression of GFAP and vimentin, among other things. This response does all kinds of other things but certainly does not leave astrocytes "normal looking" or "normal functioning". If you have to have purified morphologically and functionally similar astrocytes matrigel might be a way to go, but I would also recommend growing those matrigel astros in proximity to neurons (by perhaps growing neurons on a removable substrate separate from the astros yet incubated in the same chamber). Neuron-conditioned media is good for astrocyte development in much the same way astrocyte conditioned media is good for neuron development.

Good luck, hope this helps.

Dear david,beatriz and james... thanks alot for your answers .. yea I do agree, it would not be, so possible to get asrocytes with its intact morphology .

and now i use matri gel coated cover slip to grow the cells .

As we all know astrocytes grown in serum containing media is known to upregulte many genes than invivo situation, so i wanted to compare some of my physiological data,which i obtained from astrocytes culture, with this acutely dissociated culture , which i thought, could be more close to invivo situation

and I found this paper were they have shown acutely dissociated asrocytes with nice process baring morphology,

http://jn.physiology.org/content/84/6/2746.full

plz have a look

but i cud not gain astrocytes wit such beautiful process so far, :) ,

once again thank you very much for your time and help.

regards

shefeeq

A quick look through the methods shows on page 2747 (cell isolation) that they dice up the brain to collect hippocampi which they then take and put into cutting solution and incubate for an hour. They say this is essential for the isolation of cells with this morphology. The reason why this is essential is because this procedure induces robust reactive astrocytosis because of the damage and incubation. I don't think they say it, heck they might not even realize it, but they are looking at reactive astrocytes. Reactive astrocytes DO NOT behave like astrocytes would under normal conditions. They have altered morphologies, mobility, cell cycle, phagocytic qualities, ionic conductances, and glutamate clearance. So be weary of your interpretation of any experiments using these cells.

thank you for very informative reply James Meabon...