P0新生児ラットからの海馬ニューロンの初代培養

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Summary

解剖や個々の脳領域からの細胞の増殖は、細胞と生理的パラメータの調査を容易にします。我々は、無血清環境でのニューロンに富んだ文化を生産する主要な細胞培養のための方法を説明します。

Cite this Article

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Nunez, J. Primary Culture of Hippocampal Neurons from P0 Newborn Rats. J. Vis. Exp. (19), e895, doi:10.3791/895 (2008).

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Abstract

海馬ニューロンの生理学的特性は、一般的には、特に理由の学習と記憶における海馬の関与が、研究されています。主海馬細胞の培養は、神経科学者は、個々の細胞と単シナプスレベルでのニューロンの活性および特性を調べることができます。このビデオでは、我々は、新生児ラットから主海馬細胞を分離し、成長する方法を紹介します。海馬は2〜3分ほどの短い内の各新生児の動物から単離することが、文化は最大2週間のために維持することができます。我々はまた、簡単にレシオメトリックカルシウムイメージングのためのこれらの海馬神経細胞を使用する方法について説明します。このプロトコルは、修正なしに少しと、海馬のためのプロセスを説明しながら、それは脳の他の地域に適用することができます。

Protocol

海馬分離に先立って

海馬の分離を開始する前に、すべてのツールが無菌であることを確認してください。 70%エタノールで文化のフードを下にスプレーし、及びフード内のツールを配置します。と10cmのシャーレ、滅菌ポリ- Lコーティングされたガラス製カバースリップ、ピペッターやヒント、使い捨てピペット、および電動ピペッター - あなたは6が必要になります。この時点から、適切な無菌操作を使用することを忘れないでください。

  1. 水浴の電源を入れ、それを37℃にまで加熱されていることを確認してください
  2. 4℃で保存されている以下のソリューション° Cが必要になります。
    • 改変イーグル培地(MEM)
    • Neurobasal
    • ハンクス緩衝生理食塩溶液(HBSS)
    • ホウ酸塩緩衝液
    • ピルビン酸ナトリウム液
    • 蒸留水で滅菌濾過20%グルコース溶液

    • フードにそれらを配置する前に、70%エタノールですべてのボトルを下に散布することを確認してください。

  3. 冷凍庫から、これらの冷凍のソリューションを取ると温水浴中にそれらを配置します。
    • ウマ血清、5 mLを
    • B - 27サプリメントを1mlのアリコート
    • 100X抗生物質を1ml​​のアリコート(ペニシリンプラスストレプトマイシン)
    • L -グルタミンのと0.5mlのアリコート
  4. ソリューションが融解した後、水浴からそれらを取り出して70%エタノールでそれらを吹きかけ、そしてフードの中に置いてください。今すぐメッキとNeurobasal培地を調製することができる。
  5. ソリューションの準備が完了した後、海馬の分離を行いながらウォームアップするためにしっかりと容器にフタをし、37℃の槽の中に置いてください。
  6. また、冷凍庫の外プロテアーゼトリプシン(2.5%)のアリコートを取り、水浴に入れてください。トリプシンは、次のステップで分離されて解剖海馬を、ダイジェストになります。
  7. 、15ミリリットルコニカルチューブを取るHBSSでそれを入力し、治療群とそれにラベルを付ける。それは、孤立した海馬は、次のステップで収集されることをここにある。

海馬アイソレーション

  1. 海馬の分離を開始するには、新生児の子犬が汚れていないと、そのミルクのバンド、胎盤、およびその母親によって削除臍帯を持っていることを確認してください。
  2. きれいに、そしてボンネットに配置する70%エタノールで、ペトリ皿にスプレーして子犬を置きます。動物は、培養前に即座に安楽死させる。
  3. さらに殺菌のために一皿から子犬を取る、70%エタノールに子犬を浸し、次に滅菌HBSS中で2回洗浄に。
  4. はさみで本体からヘッドを取り外します。同じハサミを使って、皮膚や頭蓋骨を介してカット。
  5. 離れて脳から頭蓋骨をはがし、細かいピンセットを使用し、滅菌HBSSを少量含まれている小型のシャーレに脳を置く。
  6. 大脳半球バックピール。海馬は、内側側頭葉にある小さな、タツノオトシゴの形をした構造です。
  7. 15 mlのチューブにHBSS 3mlに海馬と場所を削除します。各子犬にこれらのステップを繰り返し、15 mlチューブにそれぞれの分離された海馬を置きます。今、それは単一の細胞に組織を解離する時間です。

海馬細胞解離

  1. すべての海馬が分離された後、HBSSで4.5 mlに15 mlのチューブを埋める。
  2. 水浴、エタノールでスプレー、そしてフードの場所からトリプシンを削除します。チューブにトリプシンの0.5 mlを加え、そして37℃で15分間反応させます℃に
  3. フードでは、チューブの底に沈殿している海馬を乱さないように注意しながら、滅菌ピペットでチューブからHBSS /トリプシン溶液を除去。優しくチューブと渦巻きにHBSSを5mlを追加。 ℃で5分間37℃インキュベートする。
  4. 古いHBSSを除去し、新鮮な溶液と置き換えて、二度この手順を繰り返します。
  5. 私は冷凍庫からDNアーゼのアリコートを取り出してください。 4.5ミリリットルのHBSSで海馬にDNaseの0.5 mlを追加します。 DNアーゼ、酵素不活性化を促進するために追加されます。
  6. ソリューションをひいて粉にする(または、上下にピペッティング)が均一になるまで。ホモジネートに気泡を導入しないように注意してください。
  7. トリパンブルー排除法で細胞の生存を決定する。
  8. 6から8までカバースリップを含む10cmペトリ皿を取る、とメッキの培地10mlを追加してください。カバースリップを含む皿に希望のセル数をピペ​​ット。細胞を分散し、カバースリップが重複していないことを確認して静かに渦巻く。
  9. 細胞は5%CO 2で加湿37℃インキュベーターで2〜4時間のために添付することができます。細胞は生存可能であり、接続されていることを確認した後、Neurobasal培地を含む個々の皿にカバースリップを転送する。インキュベーターに戻し、これらの皿を置きます。
  10. 週に一度、必要に応じて、新鮮なNeurobasal培地と実験的な治療で培地の3分の1を交換してください。今、培養中のこれらの細胞の機能的特性についても研究する準備が整いました。

海馬ニューロンのフラ-2カルシウムイメージング

  1. 培養海馬神経細胞が48時間(ただし、それ以前)のためにin vitroでされた後、カルシウムのイメージング実験を開始することができます。この時点で、これらのニューロンは、プロセスを拡張し始めているはず。カルシウムイメージングに最適な年齢範囲 、in vitro 3日〜7です。
  2. フラ-2と文化をロードすると、37℃で30分間午前℃で、それらがどのサンプルカルシウム指示薬染料および512ビットのCCDカメラを励起するキセノンアークランプを使用して20倍対物レンズ、すべての150msの下でイメージングすることができる後。

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Discussion

このプロトコルは、ゲイリーバンカーと金ゴズリン1の精液の仕事の修正として開発されました。この本は、培養細胞に興味を持つ人にとって不可欠な資源です - だけでなく、ニューロンに富んだ文化が現在のプロトコルで説明しています。

細胞培養における三大重要な要素は、次のとおりです。不妊、速度、使用する培地の選択。

無菌性 -層流/無菌フード、無菌条件、および滅菌インキュベーターが必要です。任意のステップで汚染だけでなく、あなたが作業している現在の実験だけでなく、計画されている可能性がその後の仕事にとって有害で​​す。インキュベーターが汚れた後は、それが洗浄と滅菌を取得するのに数週間を(または月)かかることがあります。汚染は、目に見えるレベルではすぐに明らかではないが、間違いなく細胞の生存率と細胞生理に影響を与えます。無菌性は不可欠です。

速度 -培養細胞の健康を強く細胞が大気とメディア制御された環境ではない時間の量に関連付けられています。したがって、それは細胞がメッキの媒体になるまで、動物から細胞を除去するために必要な時間が最小になることが重要です。多数のステップが変更できるか、(トリプシンでインキュベートまたは洗浄されている時間)を変更することはできませんので、脳から細胞を除去するために必要な時間です​​。多くの場合、解剖を練習。このステップの所要時間は、再現性にする必要があります。

媒体の選択 -数多くの独自のメディア、のいずれかが存在するがNeurobasalです。 Neurobasal、グリアの馴化培地を使用しての日の前に(別々に行われるために必要な-これはバンカーとゴズリン1が本の中で説明されていることは)必要とされた。関係なく、それはあなたが使用されている培地に何があるかを知ることが重要です。 Neurobasal、そのようなB - 27などのサプリメントと一緒に頻繁に使用されます。彼らは、細胞の性質に影響を与える可能性があるとして、これらの培地とサプリメントの成分が知られていることを確認してください。それは、ステロイドホルモンを(私の研究室で行われる作業の大部分はステロイドホルモンの作用を調査することを考えれば)避けるために、無血清/木炭除去/フェノールレッドを含まない培地を使用する私の研究室で標準的な方法です。このコンセプトは- あなたが使用しているソリューションで何かを知って -細胞培養のすべてのステップが重要です。

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Acknowledgments

JLNは、MH 68347によってサポートされていました。

Materials

Name Company Catalog Number Comments
Antibiotic antimitotic Invitrogen 15240062
B-27 Supplement Invitrogen 17504044 Serum free
Boric Acid Sigma-Aldrich B6768
Dnase I Sigma-Aldrich DN25
Fura-2, AM cell permeant Molecular Probes, Life Technologies F1221
Glucose Sigma-Aldrich G7528
HBSS (10X) Invitrogen 14185052
HEPES Invitrogen 15630080
L-Glutamine Sigma-Aldrich G7513
MEM Invitrogen 51200038
Neurobasal Invitrogen 12348017 Without phenol red
Poly-L-Lysine Hydrobromide Sigma-Aldrich P6282
Pyruvic Acid Sigma-Aldrich P2256
Sodium Pyruvate Sigma-Aldrich P2256
Sodium Tetraborate Sigma-Aldrich
Trypsin, 2.5% (10X) Invitrogen 15090046

DOWNLOAD MATERIALS LIST

References

  1. Banker, G., Goslin, K. Culturing Nerve Cells, 2nd Edition. The MIT Press. (1998).

Comments

45 Comments

  1. Thank you very much for this vedio. I'd want to know how to get rid of  the debris and trash after the enzyme treatment since they are always abundant in the plate and end with ruining the neurons I need.Thanks!

    Reply
    Posted by: Anonymous
    September 30, 2008 - 11:40 AM
  2. I am not sure what enzyme treatment you are refering to. Do you mean the DNase I treatment (step 5)? If you remember, the cells are first plated in plating medium (in a large dish), then moved to smaller, individual dishes that contain the Neurobasal + medium. So if you have trash and debris during the plating, that is fine - you transfer the coverslips to another dish. If you are talking about dead cells after the transfer (such as overnight) - they are usually cleaned up by the cultures themselves. Very rarely do I have any debris what-so-ever in my dishes. 

    Reply
    Posted by: Anonymous
    October 3, 2008 - 8:48 AM
  3.   Thank you very much for your reply!

    Reply
    Posted by: Anonymous
    October 6, 2008 - 8:45 AM
  4. Thank you for producing this video - I am beginning to isoalte mouse cortical and hippocampal and found this video very helpful! I have a question regarding the isolation procedure.  Our animal facility where we breed and dissect the mice, and harvest brain tissue, is about 30 min drive to the laboratory facility where we setup and maintain cultures. Given that time is a critical factor, is there a convenient stopping point in the protocol during which I can transport the cells to the lab without incurring excessive cell damage/death? Thanks very much.  

    Reply
    Posted by: Anonymous
    October 6, 2008 - 12:59 PM
  5. As a postdoctoral fellow, I too performed my tissue dissections in a different location than where we had our incubator. Through much trial and error, we found that transporting cells while in step 1 of the Hippocampal Cell Dissociation portion of the protocol (dissociated cells in 4.5ml of HBSS+) was the best. Truth be told - for us, it was a maximum of 15 minutes, transportation time. Thirty minutes may be a bit long, and you may have some cell loss. Other colleagues have placed the cells in the test tube on ice, and they have told me viability was not an issue. But I would first see if your viability is fine without the need for placing the cells on ice. Good luck.

    Reply
    Posted by: Anonymous
    October 7, 2008 - 7:59 AM
  6. Thanks very much for the advice - we'll try that, and drive faster!  ; )

    Reply
    Posted by: Anonymous
    October 8, 2008 - 10:39 AM
  7. After you remove the brain, you should put it in the cold solution that was bobbelld with O² and then place the dish on the ice. That should be ok till you get to your lab and then you can start to disect the brain in the lab.  

    Reply
    Posted by: Anonymous
    October 30, 2008 - 3:24 PM
  8. bubbled with O²? that is going to damage the the brain, oxygen radical... use mannitol in your solution...to chelate free oxygen radicals

    Reply
    Posted by: Anonymous
    September 2, 2009 - 11:10 AM
  9. Too long, try to dissect the rats in the lab

    Reply
    Posted by: Anonymous
    September 2, 2009 - 11:08 AM
  10. If the PDMS can be sterilized using UV light and how to prepare the device before using? thanks

    Reply
    Posted by: Anonymous
    October 9, 2008 - 10:48 PM
  11. First off, I do not know what you are refering to by "PDMS." Do you mean plating medium? HBSS+? Neurobasal+ media? If you mean one of those three - no, sterilizing using UV light is not always effective. We used to use UV, because it is quicker, but had issues with contamination. Buy the items sterile and only use them in a sterile hood. For the items that need to be made, sterile filter to make them sterile. And always use pressure sterilized water. And your second point - "how the prepare the device," what device? When I think of a device, I think of some tool. So I do not know what you are refering to, since no advanced tools are used in this procedure. Please respond back and I will try to answer your questions.  

    Reply
    Posted by: Anonymous
    October 11, 2008 - 10:14 PM
  12. Hi Thanks so much for this informative protocol. I just have a few questions. First, you give an extensive list of all the components needed in the particular media, but do not outline the specifc amounts/conc. of each component that make up each buffer. If you could please post the compostion of each buffer used (i.e., conc of each component in the dissection solution, plating media etc) that would be greatly appreciated! Thanks again!

    Reply
    Posted by: Anonymous
    December 2, 2008 - 4:00 PM
  13. I will do that. HBSS+: 1ml of 1M HEPES, pH 7.3 1ml of 100X antibiotic/antimitotic 10ml of 10X HBSS (calcium and magnesium free) 88ml of sterile water   Plating Medium: 86ml of MEM 10ml of horse serum 3ml of ²0% glucose (sterile filtered) 1ml of 100mM pyruvic acid   Neurobasal+: 1ml of B-²7 supplement 1ml of 100X antibiotic/antimitotic 1²5ul of L-glutamine fill to 50ml with Neurobasal (phenol red free)   I hope that is what you needed. Please let me know if you need anything more. Cheers.

    Reply
    Posted by: Anonymous
    December 5, 2008 - 8:50 AM
  14. Hi: I often have a clumping problem in my mouse hippocampal cultures. Some time after day 5-7 the neurons start getting too close and eventually the soma seem to clumb together. Do you have any idea why this is happening?

    Reply
    Posted by: Anonymous
    March 24, 2009 - 1:19 AM
  15. Clumping has never been an issue in my cultures. Two things come to mine - 1)the coating of the culture dishes/slides. We use pol-L-lysine, and the cells always stayed on well. They have to, because I do ratiometric imaging and purfuse solution across teh tops of the culltures. Maybe in your cultures, the interactive forces sticking the neurons together is stronger than that sticking the neurons to the dishes. ²) Or it could be that what you are seeing are clumps of glia. For some reason, the glial cells tend to clump. I am very sorry that I cannot be more helpful. Maybe you can send me a tiff image. That may allow for a better "diagnosis." 

    Reply
    Posted by: Anonymous
    April 19, 2009 - 10:04 AM
  16.   Dear Dr. Nunez, What is the purpose of the Acid boric? WHere do you use that? Angelo O. Rosa

    Reply
    Posted by: Anonymous
    May 21, 2009 - 9:44 AM
  17. The boric acid is used in making the borate buffer. The borate buffer is used in making the poly-L-lysine solution. Here is the recipe for the borate buffer: 1.²4g boric acid 1.90g sodium tetraborate Add both to 400ml sterile dH²0   The Poly-L-lysine is made by adding 10mg poly-L-lysine to 1ml of the borate buffer. I hope that helps.

    Reply
    Posted by: Anonymous
    May 21, 2009 - 12:11 PM
  18. when i see whether the cells have attached to the coverslip after ².5 hrs of incubation, i notice that majority of the cells do attach but on changing the focus i see that a still large number of cells remain in suspension. On transferring to new culture dish with neurobasal medium, i no longer see those large number of unattached cells, but do see quite a few cells attached to the coverslip. My question is, how do I decide whether I need to let the cells attach for more time?

    Reply
    Posted by: Anonymous
    August 7, 2009 - 6:16 PM
  19. This all depends upon the final density that you are shooting for. For both calcium imaging and Western blot analysis, ².5 hours of time in the plating medium is sufficient. If I plate for 3 hours, the density becomes a little too much for calcium imaging (I like to have some cells separate, and some cells making contacts with one another, and ².5 hours fits this purpose). But I would not go longer than 4 hours - you are probably going to have too high a density of cells. And you can keep the cells in plating medium overnight - some cells will never adhere. Probably because they are dead or just not healthy enough to adhere. But not worries - ².5-3 hours in plating medium is sufficient for most applications.

    Reply
    Posted by: Anonymous
    August 8, 2009 - 12:55 AM
  20. Thank you very much for your prompt response. As you mentioned I use them for calcium imaging and also for immunostaining after transfection, and mRNA isolation.
    Do you also do any treatment to stop the proliferation of glia cells?

    Reply
    Posted by: Anonymous
    August 8, 2009 - 4:27 PM
  21. I use ara-C (cytosine arabinoside) to stop unwanted glial cell proliferation.

    Reply
    Posted by: Anonymous
    August 11, 2009 - 6:46 AM
  22. Dear Dr Nunez,
    What are the differences of a cell culture and a slice hippocampal culture will have on the tissues? Will neurons developed differently? Will neurons grow differently in a cell culture, compared to a slice tissue culture of hippocampus.?DŒs the structure of the hippocampus has any significant impact on neurogenesis and neurons development, compared to cell culture methodology? Thank you.

    Reply
    Posted by: Anonymous
    September 2, 2009 - 11:17 AM
  23. Cell culture has dispersed cells, while the slice culture keeps the connectivity within the hippocampus. Basically, the cell culture is ripping the hippocampus apart, and plating the individual cells, while the slice culture removes the hippocampus as a slice and grows that whole thing in a dish. Yes, neurons will develop differently between the methods - in cell culture, they grow in the absence of their normal neighbors. If you can, I would suggest slice cultures for looking at normal development and neurogenesis. But if you want to see individual cell properties (independent of neighbors), do the cell cultures. I am sure hippocampal development is more altered in the cell cultures. We do not see much neurogenesis in cell culture, but we do see the normal developmental events in the individual neurons (such as GABA-mediated excitation). Both are great tools, and both have their strengths. I hope that helps. Contact me again if you would like further advice.

    Reply
    Posted by: Anonymous
    September 4, 2009 - 9:13 AM
  24. It is really useful to have this video at hand, thank you very much. I wanted to ask about a matter not discussed here.
    I am new to primary neuronal cell cultures and I am interested in buying a CO² incubator. How important is the choice of incubator for the culturing of neurons, any thoughts/advice on the subject? It seems that IR sensor is a must, but when it comes to air jacket vs water jacket, fan no fan, UV vs high temperature decontamination I am lost. Any advice on models/reliable companies would be highly appreciated! Thank you in advance!

    Reply
    Posted by: Anonymous
    June 25, 2010 - 3:55 PM
  25. I know it has been a long time since your response - my apologies. Air vs water jacketed is important in the regulation of temperature. The assumption being that water maintains the temp more efficiently than air. The fan is another item that helps with temp maintenance, especially when you are opening and closing the door to the incubator often. I think a fan is a must, and would prefer water jacketed. I know that UV is more effective - we use UV decontamination in our hoods, and the hood people have never told us that high temps are 100% effective. As for model, I love the VWR I have used. I know that you can spend an arm and a leg. But VWR has been great for us.

    Reply
    Posted by: Anonymous
    March 2, 2012 - 9:36 PM
  26. Thank you very much for this vedio. I am wondering if I know you measure osmolarity of plating media and feeding media?

    Reply
    Posted by: Anonymous
    November 25, 2010 - 1:25 PM
  27. I am sorry, but I have never measured the osmolarity of the plating medium or the culture medium.

    Reply
    Posted by: Anonymous
    November 30, 2010 - 3:12 PM
  28. DR,Joseph.How to avoid the growth of glial cell.Could you tell me how to use ara-C?I was confused.thanks

    Reply
    Posted by: Anonymous
    November 27, 2010 - 1:52 AM
  29. The ara-C, also called cytosine arabinoside, controls glial cell proliferation. This drug affects dividing cells, and since glial cells are really the only thing dividing, it is potent in killing any dividing glial cells. The ara-C can be added after cells have been in culture. Please let me know as to the confusion - do you mean concentration? Vehicle for the ara-C? How often to administer?

    Reply
    Posted by: Anonymous
    November 30, 2010 - 3:18 PM
  30. Thank you for the video. I would like to know protocol to make PDL coated coverslip or dishes. Thanks

    Reply
    Posted by: Anonymous
    January 7, 2011 - 3:35 PM
  31. First rinse the coverslips in dH²O and leave them in nitric acid overnight. Then dip two times in milliQ H²O, and dry in an oven set to 40 degrees. Autoclave the coverslips and allow to cool to room temp. All of the remaining procedures should be performed in a laminar flow hood. Place 4-5 coverslips in a 100mm petri dish. Combine 0.5ml 10X poly-l-lysine and 4.5ml borate buffer. Cover each coverslip with 3-4 drops of the poly-l-lysine/borate buffer solution. place the coverslips overnight in a 37degree C incubator. Rinse twice with sterile H²O. If not using immediately, cover the petri dish with parafilm and store in the fridge for up to one month.

    Reply
    Posted by: Anonymous
    March 2, 2012 - 9:42 PM
  32. HiA²81;
    Thank you very much for your vedio. Neurons initially grow well, but begin to detach and be broken into pieces after day ²-4; no cell survive past 1 week. Do you have any idea why this is happening? Thanks!

    Reply
    Posted by: Anonymous
    May 4, 2011 - 5:45 AM
  33. My guess is that it is a problem with the poly-L coating of the coverslips. You may want to try fresh poly-L, or another method (some people use poly-D-lysine). I have always had survival of at least 10-14 days.

    Reply
    Posted by: Anonymous
    May 4, 2011 - 12:18 PM
  34. Thank you for your reply, maybe I can change the time of coating.

    Reply
    Posted by: Anonymous
    May 6, 2011 - 10:02 AM
  35. What do you mean - change the time of the coating? You mean coat for longer? Or use "fresher" coverslips?

    Reply
    Posted by: Anonymous
    March 2, 2012 - 9:43 PM
  36. Dr. Joseph Nunez, there are several comments regarding you use of Ara-C to control glial growth in your cultures. I have several questions. When do you first treat your cultures with Ara-C? Do you continue to treat with additional Ara-C and how often? What concentration do you use? I have been trying to use Ara-C in my own cultures but this is resulting in extensive debris. Do you have any further suggestions? Thanks!

    Reply
    Posted by: Anonymous
    May 23, 2011 - 3:57 PM
  37. Honestly, I try to avoid using Ara-C as much as I can because of the debris. I use it 4 days after the day of culturing. I use a concentration of 1uM. I do not continue to use it given I only use my cultures for a maximum of 14 days. My suggestion (please don't take this the wrong way) is to make my initial culturing as glial free as possible. So culturing at an earlier embryonic age, using less steroid containing growth factors, etc, has helped me. Sorry I cannot be more helpful.

    Reply
    Posted by: Anonymous
    March 2, 2012 - 9:48 PM
  38. Hi, Thank you very much for the video. I am wondering if I know how you make a DNase solution from the powder?

    Reply
    Posted by: Anonymous
    October 6, 2011 - 11:10 AM
  39. add one vial of DNA powder to 500ul ddH²O

    Reply
    Posted by: Anonymous
    March 2, 2012 - 9:45 PM
  40. Hi, Thank you very much for the video. I am wondering if I know how you make a DNase solution from the powder?

    Reply
    Posted by: Anonymous
    October 6, 2011 - 11:10 AM
  41. Hello,
    A question completely unrelated to the video. Hope you could help.
    I've just begun neuronal culture experiments with chick embryos.
    I isolated the forebrain portion of the embryo from 8-day old eggs, performed a primary neuron culture isolation procedure and have counted the number of viable cells.
    I would like to know, how I would test the efficiency of my protocol/ the neuronal culture done.
    Please do let me know. Any sort of structural recognition I should be doing? How do I go about the same?
    Thanks.
    P.S: Thank you for the video.

    Reply
    Posted by: Anonymous
    October 13, 2011 - 12:30 AM
  42. Not sure what you mean by efficiency, but in my lab we also are culturing CFNs from E8 embryos. You can always compare numbers of trypan blue + versus - neurons on the hemacytometer to get a % of viable cells. A very good prep will get you about 10^7 viable
    neurons per ² chick telencephali hemispheres

    Reply
    Posted by: Anonymous
    January 17, 2012 - 10:36 PM
  43. Dr. Joseph Nunez, glad to see you again, I²16;ve solved my problem. Actually, the death of my cells was caused by dissociation. Maybe the dissociation is not enough, so I pipetted up and down so strongly,and
    this may hurt the cell.

    Reply
    Posted by: Anonymous
    March 5, 2012 - 12:39 AM
  44. A very helpful video protocol. One question, is it ok if we cut the heads of the pups and bring them to culture room. This kind of transportation will take 15 minute, and then isolate the hipocampus for cell or tissue culturing. Instead of isolating the hippocampus straight after choppiing head in the animal facility.

    Reply
    Posted by: Farah S.
    August 21, 2012 - 4:55 AM
  45. What you mentioned was how I (as a postdoc) used to culture when the location of our animal colony, lab and culture room were far from one another. While the best choice is to have the shortest time interval between euthanasia/brain removal and culturing, we had consistently high numbers of viable cells with the method you suggest.

    Reply
    Posted by: Joseph N.
    August 22, 2012 - 10:26 AM

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