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Culture of rat cortical neurons - E16 or E18? (Archived)


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#1 bioforum

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Posted 03 February 2009 - 11:15 PM

Culture of rat cortical neurons - E16 or E18? (May/15/2007 )

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Hi,

I´m confused since I have to do a culture of rat cortical neurons and I´ve read that some people use embryos at E16 stage while others use them at E18. I´ve asked people who use them at E16 and they told me that at that stage they obtain a more pure neuronal culture because at E18 they obtained more astrocytes mixed with neurons.

Also, E16 embryos are very little and their brains a bit difficult to handle, and very soft.

What are your opinions? What stage should I use? :huh:

thanks

-Pumuki-



nobody knows? :)
:(

-Pumuki-


Yes, definitely E18 is better as it is too hard to handel the head of the fetus to remove the brain at E16. Additionally there is not many astrocytes at E18 (I think it is just about 20%); furthermore if you want to work on neurons you can culture the cells in neurobasal medium + B27 (instead of serum).
Hope this helps,
Good luck.

-saya-



i am doing cortical neuron culture in mice. E-16 is my preferred as astrocytes increase with age. With E-16, we get quite a bit of astrocytes so one can imagine the number of astrocytes in E-18. We actually have to use antimitotic agents to kill the non-neuronal cells.

Lets say you start with even 5% of non-neuronal cells in the beginning, they multiple and would utilize most of the nutrients from, the media for the neurons. Thats why one has to choose the right age and make sure the non-neuronal cells are kept to a minimum.

Keep the pups in ice cold HBSS and also dissect the brains in cold HBSS, this helps so that tissue is not soft. Also you need practice to get it going smoothly.

Good Luck !!!

-scolix-


Thanks

<!--quoteo(post=98639:date=May 21 2007, 10:32 PM:name=saya)-->

QUOTE (saya @ May 21 2007, 10:32 PM)
<!--quotec-->Yes, definitely E18 is better as it is too hard to handel the head of the fetus to remove the brain at E16. Additionally there is not many astrocytes at E18 (I think it is just about 20%); furthermore if you want to work on neurons you can culture the cells in neurobasal medium + B27 (instead of serum).
Hope this helps,
Good luck.<!--QuoteEnd-->
<!--QuoteEEnd-->

Does neurobasal medium (from Gibco??) + B27 inhibit glia proliferation? I mean, does it include an antimitotic agent?

One colleague uses E18 rat embryos to obtain a mixed culture, that is, both astrocytes and neurons at the same time and even in this case also uses AraC to avoid excesive glia proliferation. And when she wants neurons she uses E16 and a higher concentration of AraC.

On the opposite side, another girl says that she obtains just 5% astrocytes when using E18... I can´t understand such a big difference :)

<!--quoteo(post=98645:date=May 21 2007, 11:38 PM:name=scolix)-->
QUOTE (scolix @ May 21 2007, 11:38 PM)
<!--quotec-->i am doing cortical neuron culture in mice. E-16 is my preferred as astrocytes increase with age. With E-16, we get quite a bit of astrocytes so one can imagine the number of astrocytes in E-18. We actually have to use antimitotic agents to kill the non-neuronal cells.

Lets say you start with even 5% of non-neuronal cells in the beginning, they multiple and would utilize most of the nutrients from, the media for the neurons. Thats why one has to choose the right age and make sure the non-neuronal cells are kept to a minimum.

Keep the pups in ice cold HBSS and also dissect the brains in cold HBSS, this helps so that tissue is not soft. Also you need practice to get it going smoothly.

Good Luck !!!<!--QuoteEnd-->
<!--QuoteEEnd-->

Ok, so you two don´t agree about the stage either :huh:
Yes, people told me that rat brains at E16 are very small and sometimes difficult to handle... so you scolix are a real hero, working at E16 with mice and their tiny skulls :mellow: As you say, some colleagues told me that they use AraC in order to avoid glia proliferation, because glia grows quite fast so neurons run out of nutrients.

I removed cortex just once, and it was quite difficult to remove meninges (which have plenty of red vessels, haven´t they?) without damaging the brain

I think I will try both at E16 and E18, and do an ICH and see the % of astrocytes and neurons I obtain in both cases.

-Pumuki-



Inorder to remove the meninges, I suggest using 2 forceps and one could actually peel the meninges from half a brain (hemisphere) at once. The extent of astrocytes is difficult to assess. try the ICH and you should haave a good idea. But try the ICH on different days as the nonneuronal cells do divide.

We use antimitotics on day 5 and change media on day 7 and then leave the cultures for upto 4 weeks. The non neuronal cells are mosly dead but they are still there. Its better they are also present as the neurons in the brain are surrounded by glial cells all the time. so I fell its not fair to completely remove the glial cells.


E-16 mice is not bad to try. Practice will help you. Keep practicing.

good luck !!!

-scolix-


<!--quoteo(post=98777:date=May 22 2007, 06:49 PM:name=scolix)-->

QUOTE (scolix @ May 22 2007, 06:49 PM)
<!--quotec-->Inorder to remove the meninges, I suggest using 2 forceps and one could actually peel the meninges from half a brain (hemisphere) at once. The extent of astrocytes is difficult to assess. try the ICH and you should haave a good idea. But try the ICH on different days as the nonneuronal cells do divide.


We use antimitotics on day 5 and change media on day 7 and then leave the cultures for upto 4 weeks. The non neuronal cells are mosly dead but they are still there. Its better they are also present as the neurons in the brain are surrounded by glial cells all the time. so I fell its not fair to completely remove the glial cells.


E-16 mice is not bad to try. Practice will help you. Keep practicing.

good luck !!!<!--QuoteEnd-->
<!--QuoteEEnd-->

Thank you very much!!

-Pumuki-



ooops, new question: What do you prefer to disgregate cells: trypsin (some people says it damages cells too much) or mechanically by pipetting repeatedly through a fire-polished pipette... :P
thanks again

-Pumuki-


<!--quoteo(post=98920:date=May 23 2007, 04:17 AM:name=Pumuki)-->

QUOTE (Pumuki @ May 23 2007, 04:17 AM)
<!--quotec-->ooops, new question: What do you prefer to disgregate cells: trypsin (some people says it damages cells too much) or mechanically by pipetting repeatedly through a fire-polished pipette... :P
thanks again<!--QuoteEnd-->
<!--QuoteEEnd-->


we use trypsin for 15 min. then add DNase to degarde DNA and then using fire polished pipette to gently disassociate the tissue.

-scolix-



Yes, neurobasal medium can inhibit astrocytes growth (you can see this:
http://www3.interscience.wiley.com/cgi-bin...=1&SRETRY=0
To my experience trypsin is so important for cortical cell culture and I just use trypsin without any DNase usage.

-saya-


Thanks!
Saya, the paper is very complete, really good :)

Finally, yesterday we had a discussion in our lab: some people said that the pregnant rat should be very quickly killed by cervical dislocation or guillotine without being anesthetized :o :o :o :o (I suppose this wouldn´t be approved by an ethical comittee...) because anesthesia would damage embryonic neurons and reduce viability, while others said that it must be killed by an overdose of anesthesia.
Furthermore, in the case of anesthesia, they also didn´t agree because some people thinks that i.p. anesthesia (pentobarbital) causes more damage to the embryos (or, at least, is quicker) that the inhaled one (halothane).

what do you think? :(

-Pumuki-




<!--quoteo(post=99059:date=May 24 2007, 12:59 AM:name=Pumuki)-->

QUOTE (Pumuki @ May 24 2007, 12:59 AM)
<!--quotec-->Thanks!
Saya, the paper is very complete, really good :)

Finally, yesterday we had a discussion in our lab: some people said that the pregnant rat should be very quickly killed by cervical dislocation or guillotine without being anesthetized :o :o :o :o (I suppose this wouldn´t be approved by an ethical comittee...) because anesthesia would damage embryonic neurons and reduce viability, while others said that it must be killed by an overdose of anesthesia.
Furthermore, in the case of anesthesia, they also didn´t agree because some people thinks that i.p. anesthesia (pentobarbital) causes more damage to the embryos (or, at least, is quicker) that the inhaled one (halothane).

what do you think? :(<!--QuoteEnd-->
<!--QuoteEEnd-->

We anesthesize pregnant rats by CO2. If rats remained more in CO2 chamber it can kills the rat because of deep anesthesia and therefore no cervical dislocation is needed. Personally I prefer to anesthesize the rat with CO2 and then do cervical dislocation. It workes fine.

-saya-



<!--quoteo(post=99094:date=May 24 2007, 03:57 PM:name=saya)-->

QUOTE (saya @ May 24 2007, 03:57 PM)
<!--quotec-->We anesthesize pregnant rats by CO2. If rats remained more in CO2 chamber it can kills the rat because of deep anesthesia and therefore no cervical dislocation is needed. Personally I prefer to anesthesize the rat with CO2 and then do cervical dislocation. It workes fine.<!--QuoteEnd-->
<!--QuoteEEnd-->

That´s a good option. thank you :)

-Pumuki-


We used to kill animals by CO2 in my old lab. Now, we just kill it by isofluorane and then by cervical dislocation before opening the abdomen.

-scolix-


<!--quoteo(post=99130:date=May 24 2007, 09:06 PM:name=scolix)-->

QUOTE (scolix @ May 24 2007, 09:06 PM)
<!--quotec-->We used to kill animals by CO2 in my old lab. Now, we just kill it by isofluorane and then by cervical dislocation before opening the abdomen.<!--QuoteEnd-->
<!--QuoteEEnd-->

ok,so it seems there is no problem for neurons if you kill with anesthesia.
Thanks scolix

-Pumuki-



#2 sharma

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Posted 03 August 2009 - 07:47 AM

Hello,
i am having the same probelm with my cortical cells and i did get good ideas readig everybody's scraps through this forum. I have been working on E18 using 10% Mitotic Inhibitors and making sure that i extract my cortical tissues in ice cold HBSS. Everything looks good until 7 days after plating. However, after that the number of dead cells keep increasing. I tried using Mitotic inhibitor twice (day 2nd and day 9) after plating. However, the number of dead cells do not decrease. Wonder what i can do because i need to keep these guys healthy until day 14 to do my experiments..:( i use SMEM + Horse Serum as my media + trypsinize my cells +dissociate them using fire polished pipette :( :(
Sharma

#3 pretygirl

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Posted 07 August 2009 - 05:31 PM

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