So far I have used the neurosphere assay for my experiments but due to many drawbacks of this assay I would rather use a monolayer cell culture of neural stem cells. Has anyone successfully grown a monolayer of primary neural stem cells? If so, what are the potential problems (e.g. spontaneous differentiation, etc.) and how to avoid them?
I have tried playing around with monolayer derived from the SVZ NSC. Look at the attached protocol. It works really well. I would recommend growing neurospheres first, maybe take tertiary spheres dissociate them, then grow them in monolayers. This would get rid of any contaminating tissue from the dissection. Also, use can use Accutase for dissociation, it gentler on the cells, and you do not need to go through preparing the enzymes for dissociation before hand. One caveat is you will use a lot of GF for this assay, which might affect cell behavior. I have not seen spontaneous differentiation with the monolayers, it did not see any neurons at 3 days differentiation. Good luck
I have tried playing around with monolayer derived from the SVZ NSC. Look at the attached protocol. It works really well. I would recommend growing neurospheres first, maybe take tertiary spheres dissociate them, then grow them in monolayers. This would get rid of any contaminating tissue from the dissection. Also, use can use Accutase for dissociation, it gentler on the cells, and you do not need to go through preparing the enzymes for dissociation before hand. One caveat is you will use a lot of GF for this assay, which might affect cell behavior. I have not seen spontaneous differentiation with the monolayers, it did not see any neurons at 3 days differentiation. Good luck
This is excellent, just what I was looking for! Thank you very much! At the moment, for setting up the neurosphere culture I am using a mixture of diaspase II and papain solution dissolved in HBSS. I have heard it is even better than Accutase and so far it works beautifully so I might try to use it for this assay too. Can I just ask you what markers did you use for immunolabelling your colonies? The typical Nestin, Sox2 for example? Did you maybe try the BLBP by any chance?
I tried IF for this assay once after differentiation, and it worked fine. My aim was to see when is the earliest time I can detect neurons, so I stained my differentiating cultures at 3d, but there were very few neurons then. I used GFAP for astrocytes, Tuj1 for neurons. We typically use O4 for oligodendrocytes. I have not tried using Sox2, BLBP or nestin. Nestin should work fine, but it is not specific to stem cells in vivo, it labels some TAPs and neuroblasts as well. Have you tried BLBP for f-IHC on sections?
Oh ok. I just tried to show that the culture of the cells I have got is in fact a stem cell culture, hence I tried the stem cell markers. Some markers worked better than others and I am struggling to detect Sox2 at the moment, but it might be due to antibody I use. Do you differentiate your cells on glass coverslips? If so, what do you coat them with? Is it PDL? I personally have not used BLBP on sections, but some people from my lab have and it worked well. I was told that they were cryostat, rather thin sections. Are you interested in BLBP?
The monolayers adhere quite well to the tissue culture treated plates (Falcon). I have not tried having them adhere on any substrate. You can differentiate the cells in the plates by just changing to the differentiation medium. I want to test out the BLBP Ab, do you know a good Ab?
I will attach a paper here, we used the BLBP here on sections and it worker rather well. We have not tried any other antibody, so I guess my exerience is limited. Hope it works well for you too.
we are having issues in growing the stem/progenitors as neurosphere in culture from postnatal day 5 old BLK6 mice brain, they are grown for 2 days, then they start disintegrating into single cells. I have changed the B27 supplemented DMEM:F12 media, we use Glutamax as glutamine supplement, EGF & FGF2 as growth factors and gentamycin.Please provide some help.
Ekta, are you adding heparin to the media to stabilize the growth factors? I found that it does make a difference. I also advise the neurobasal media, you can try if that would help
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