Could you tell cell culture procedure a bit more, since would be easy to suggest some solutions.

Hi Sivaraj,

So i used a neuro progenitor population which was cultured for over 80 days now in media with 1% FBS. I have a mixed population of more glial/astrocyte progenitors and weird pericyte like cells (big expanded cell, large nucleus) and very little neurons. I want to selectively enrich for the astrocytes, but was not successful in my sorting. I am looking for other methods to enrich for astrocytes. 

Hi Janani,

which enzymes have you used before labeling with the GLAST beads? This is a crucial step. Have you worked with the NTDK-T?

Have you tried ACSA-2 MicroBeads as an alternative for astrocyte isolation? This is a direct conjugate (antibody-beads) so a faster protocol , and ACSA-2 can be used with papain and the NTDK-P in general performs a little bit better than the NTDK-T.

ACSA-2 recognizes all the GLAST positive cells and a few more weakly GLAST positive astrocytes. It's absolutely astrocyte-specific.

In any case working with titrated and consistent antibody-beads conjugates leads to more reproducible results than immopanning which involves new coating , so a new, antibody-dish "conjugation" for each experiment.

Please let me or our excellent TecSupport team know if you need more advice.

Sandra

Oh sorry, I missed the word human. So then GLAST is the right choice. ACSA-2 is only for mouse.  How old is the tissue that you start with?

you can try shaking the culture at 120rpm for 8 -12hrs.

Through shaking I am sure you can get rid off pericyte structured cells and possibly also from neurons.

still if you get contamination of neurons I would suggest you to go isolation of astrocytes by density gradient centrifugation method.

Hi Sivaraj,

Many thanks for your suggestions. Could you elaborate on the shaking method? When should i shake the culture- at what confluency or under what treatment? 

Hi Sandra,

Thanks for your inputs as well. I used life tech EAAT1 antibody with dynabeads but that didnt work. I have not tried MACs GLAST kit because i dont have the stand and separator. I am trying to get in touch with the UK rep to get more info. 

I had a bad experience with immunopanning - in my hands I could not get a good purity with mouse cells. Protocol is for murine cells max till 20 days old - so no option for adult tissue from human. Also protocol is quite long and performed at room temp - not sure if cells change during this procedure. I think GLAST-microbeads from miltenyi or FACS with GLT-1 (papain-stable) or GLAST (papain-sensitive, trypsin-stable) are the best options for human cells.

The study below might be quite useful to check for immunopanning strategies for human astrocytes:

"Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse" by Zhang Y et al

Neuron. 2016 Jan 6;89(1):37-53. doi: 10.1016/j.neuron.2015.11.013.