Hi, I have been trying to induce neural induction in my mESC lines by using -DMEM-F12 + N2+B27+NEAA+ bFGF. Although I started getting rosette formation from day5/6 onward, and mature neural marker expression from day7 onward, I also had lots of other lineage (mesodermal/ endodermal) contamination. I performed this NI for a period of 14 days, changing media everyday.
To counter the other lineage contamination, I started with a different protocol using small molecule inhibitors using the following media cocktail- Neurobasal media+DMEM-F12+NEAA+Glutamax+beta-mercaptoethanol+insulin+ B27+N2+bFGF+SB+LDN. Although this protocol has worked well in the hands of another researcher using mESC HM1, it is not working at all with my lab's established mESC lines. Even until the day 8 of N.induction, I can't morphologically see any neural lineage appearance. I just came across this publication: http://www.sciencedirect.com/science/article/pii/S0898656814001971 which discusses the role of SB in inhibiting differentiation and maintaining pluripotency of mESCs.
I am quite confused at this point, and would highly appreciate if someone could shed some light on these issues I am facing. I have cells in culture in both conditions right now, I am tempted to take out SB in some wells and see the effect, but it would be great to get some advice before I go manipulating the protocols.
Many thanks.
Regards,
Pooja.
Hello
You have a good protocol , also read protocols here
will help you
Good Luck
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.558.1404&rep=rep1&type=pdf
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0006286
Hello Pooja,
Truly speaking, you have made your second protocol quite complicated. First protocol is good but I will suggest some changes. N2 supplement is known to favour the expression of neural genes and inhibits the growth of non-neuronal cells in the culture (or any undifferentiated cells). You can use N2 supplement (1%), add G5 supplement with it to increase the efficacy. Use neurobasal media and there is no need to use DMEM+HAM F12 , NBM is sufficient for this purpose. Please remove excessive things from your supplements like insulin, BME etc. as they can support growth of nonspecific cells. Use 2% KSR to replace FBS. Please give your updates about the same.
Hope it helps !
Hello Pooja,
Here is a Research paper attached from the work conducted by one of the previous student in my lab which was quite successful. You can also find the references for the selected protocol which could further help you.
Hope this helps.
Good Luck!
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