P0 신생아 쥐에서 Hippocampal 신경의 주요 문화

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Summary

해부 및 개별 뇌 영역에서 세포의 성장은 세포와 생리적 매개 변수의 조사를 용이하게합니다. 우리는 혈청이없는 환경에서 신경 세포 - 풍부한 문화를 생산하는 주요 세포 culturing에 대한 방법을 설명합니다.

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

hippocampal 신경의 생리적 특성은 일반적으로 특히 때문에 학습 및 메모리에 해마의 참여의 조사입니다. 기본 hippocampal 세포 culturing은 neuroscientists는 개별 세포와 단일 버렸네 수준에서 뉴런의 활동과 속성을 확인하실 수 있습니다. 이 비디오에서는, 우리는 신생아 쥐의 기본 hippocampal 세포를 분리하고 성장하는 방법을 보여줍니다. 해마는 2~3분만큼 짧은 각 신생아 동물로부터 격리 수 있으며, 문화 두 주에 유지하실 수 있습니다. 우리는 또한 간략하게 ratiometric 칼슘 이미징 이러한 hippocampal 뉴런을 사용하는 방법을 보여줍니다 것입니다. 이 프로토콜은 어떠한 수정을 거의, 해마에 대한 과정을 설명하는 동안, 그것은 두뇌의 다른 지역에 적용할 수 있습니다.

Protocol

hippocampal 분리하기 전에

hippocampal 고립을 시작하기 전에 모든 도구 멸균되어 있는지 확인합니다. 70 % 에탄올과 문화 후드를 스프레이하고, 후드 내부의 도구를 놓으십시오. , 멸균 폴리 - L 코팅 유리 coverslips, pipettors 및 팁, 일회용 pipettes, 그리고 전기 pipettor과 10 cm 배양 접시 - 당신이 6 필요합니다. 이 시점부터는 올바른 살균 기술을 사용해야합니다.

  1. 물 목욕 켜고 그것이 37 ° C.까지 가열되어 있는지 확인합니다
  2. 4 저장되어있는 다음과 같은 솔루션 ° C가 필요합니다 :
    • 수정된 이글의 중간 (멤)
    • Neurobasal
    • 행크의 버퍼 생리 식염수 (HBSS)
    • Borate 버퍼 솔루션
    • 나트륨 pyruvate 솔루션
    • 증류수에 멸균 필터링 20 % 포도당 용액

    • 후드에서 그들을 배치하기 전에 70 %의 에탄올과 함께 모든 병을을 스프레이하십시오.

  3. 냉동실에서 이러한 냉동 솔루션을 가지고 온수 물 목욕에서 그들을 장소 :
    • 말 혈청 5 ML의 나누어지는
    • B - 27 보충의 한 ML의 나누어지는
    • 100X 항생제의 한 ML의 나누어지는 (페니실린 스트렙토 마이신 플러스)
    • L - 글루타민 및 0.5 ML의 나누어지는
  4. 솔루션은 해동 후, 물 목욕 그들을 데리고 70 % 에탄올과 그들을 스프레이하고, 후드에서 그들을 놓으십시오. 이제 도금과 Neurobasal 매체 준비하실 수 있습니다.
  5. 해결책이 준비 후 hippocampal 고립을 수행하는 동안 워밍업을 단단히 용기 뚜껑과 37 ° C 목욕에서 그들을 놓으십시오.
  6. 또한, 냉장고 밖으로 프로 테아제 트립신 (2.5 %)의 나누어지는를 타고 물 욕조에 넣습니다. 트립신은 다음 단계에서 격리됩니다 해부하는 해마를 소화합니다.
  7. , 15ml 원뿔 튜브를 타고 HBSS로 기입하고, 치료 그룹으로 분류. 그것은 고립된 hippocampi는 다음 단계에서 수집됩니다 여기에 있습니다.

Hippocampal 절연

  1. hippocampal 격리를 시작하려면 신생아 새끼는 깨끗해하고 우유 밴드, placentas, 그들의 어머니에 의해 제거 탯줄 코드를 적이 있는지 확인하십시오.
  2. 청소하고, 후드에서 그들을 곳으로 70 %의 에탄올과 배양 접시에있는 스프레이 새끼를 놓습니다. 동물은 euthanized culturing 바로 앞에 있습니다.
  3. 자세한 살균 한 접시에서 강아지를 타고 70 % 에탄올로 강아지를 찍어 다음 멸균 HBSS 두 씻는다로.
  4. 가위로 시체에서 머리를 제거합니다. 같은 가위를 사용하여 피부와 두개골을 통해 했네요.
  5. 멀리 두뇌에서 두개골 껍질, 괜찮아요 족집게 한 켤레를 사용하여 멸균 HBSS의 작은 금액을 포함하는 작은 페트리 접시에 머리를 놓으십시오.
  6. 대뇌 반구 다시 필. 해마는 중간 측두엽에있는 작은 해마 모양의 구조입니다.
  7. 15 ML 튜브의 HBSS 3 ML에 해마과 장소를 제거합니다. 각각의 강아지와 함께 이러한 단계를 반복하고, 15 ML 튜브에 각각 분리된 해마를 놓으십시오. 이제 하나의 세포로 조직을 떼어 놓다 시간이 있습니다.

Hippocampal 세포 분리

  1. 모든 hippocampi가 고립되어 후 HBSS와 4.5 ML로 15 ML 튜브를 입력하십시오.
  2. 후드에있는 물 목욕, 에탄올과 스프레이, 장소에서 트립신을 제거합니다. 튜브에 트립신 0.5 ML를 추가하고 37 15 분 동안 품어 ° C.
  3. 후드에서 튜브의 바닥에 정착 hippocampi을 방해하지 않도록주의하고, 멸균 피펫으로 튜브에서 HBSS / 트립신 용액을 제거합니다. 부드럽게 관과 소용돌이에 HBSS 5 ML을 추가합니다. ° C 5 분 37 알을 품다.
  4. 이전 HBSS를 제거하고 신선한 솔루션으로 대체 두번이 단계를 반복합니다.
  5. 냉동고의 I가 DNAse의 나누어지는 가져가라. 4.5 ML의 HBSS에 hippocampi에 DNase 0.5 ML를 추가합니다. DNAse는 효소 불활 성화를 촉진에 추가됩니다.
  6. 솔루션을 씹다 (또는 그리고 아래로 피펫)는 동질 때까지. homogenate에 거품을 소개하지 않도록주의하십시오.
  7. trypan 블루 제외 메서드를 사용하여 세포 생존을 확인합니다.
  8. 6-8 coverslips을 포함 10cm 페트리 접시를 타고, 도금 매체 10 ML를 추가합니다. coverslips 들어있는 접시에 세포의 원하는 번호를 피펫. 소용돌이는 부드럽게 세포를 분산하고, coverslips가 중복되지 않도록합니다.
  9. 세포 5 % CO 2 humidified 37 ° C 배양기에서 2-4시간 위해 첨부할 수 있습니다. 세포가 가능한되며 첨부 것을 확인한 후, Neurobasal 매체를 포함하는 각각의 요리에 coverslips을 전송하기만하면됩니다. 인큐베이터에 다시 이런 요리를 놓습니다.
  10. 일주일에 한 번, 필요한만큼, 신선한 Neurobasal 매체와 실험 치료와 매체의 3 분의 1을 교체하십시오. 이제 문화에서 이러한 세포의 기능 특성 연구로 준비가되어 있습니다.

Hippocampal 뉴런의 Fura - 2 칼슘 이미징

  1. 교양 hippocampal 뉴런 48 시간 (하지만 이전)에 대한 체외에 있었 후, 칼슘 이미징 실험을 시작할 수 있습니다. 이 시점에서,이 뉴런들은 프로세스를 확장하기 시작한다. 칼슘 이미징을위한 최적의 연령 범위는 체외 3 일에서 7입니다.
  2. Fura - 2와 부하 문화 37 30 분 AM ° 그들이 어떤 샘플 매 150ms를 칼슘 표시기 염료 및 512 비트 CCD 카메라를 자극하는 제논 아크 램프를 사용하여 20X 목적에 따라 몇 군데 수있는 후에 C.

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Discussion

이 프로토콜은 게리 뱅커와 김 고슬린 1의 혁신적인 작품의 수정으로 개발되었습니다. 이 책은 culturing 세포에 관심이 사람을위한 필수적인 리소스입니다 -뿐만 아니라 신경 세포 - 풍부한 문화는 현재 프로토콜 설명했다.

세포 culturing에있는 세 개의 가장 중요한 요소는 다음과 같습니다 불임, 속도, 사용되는 매체의 선택이다.

불임 - 층류 / 살균 후드, 무균 조건 및 무균 인큐베이터가 필요합니다. 모든 단계에서 오염뿐만 아니라 당신이 작업하고있는 현재의 실험뿐만 아니라, 계획되었을 수도 후속 작업 해로운 것입니다. 인큐베이터가 오염 된 후에, 그것이 세척 및 멸균하려면 주 (또는 달)이 걸릴 수 있습니다. 오염이 눈에 띄는 수준에서 즉시 분명 없지만, 확실히 세포 생존과 세포 생리학에 영향을 미칠 것입니다. 불임은 필수적입니다.

속도 - 교양 세포의 건강은 강력하게 세포의 분위기와 미디어 환경을 통제할 수없는 시간의 금액에 묶여있다. 따라서, 그것은 세포 도금 매체에까지 동물의 세포를 제거하는 데 필요한 시간의 양이 최소한 것을 중요합니다. 수많은 단계 (트립신의 잠복기 또는 세차되고 시간) 변경될 수 없기 때문에, 어떻게 변경할 수 있습니다하면 두뇌의 세포를 제거하는 데 필요한 시간의 금액입니다. 자주 절개를 연습합니다. 이 단계에 필요한 시간의 양은 다시 재현할 수 있어야합니다.

매체의 선택 - 수많은 독점적인 매체의 하나가있다는 Neurobasal입니다. Neurobasal, glial 시설 매체 사용의 일 이전에 (별도 만들 필요 - 이것은 뱅커와 고슬린 1 책에서 설명하는 것은)가 필요했습니다. 상관없이, 당신이 사용중인 매체에 뭔지는 알고하는 것이 중요합니다. Neurobasal 등 B - 27와 같은 보완과 함께 자주 사용됩니다. 그들은 세포의 속성에 영향을 미칠 수있는 이러한 매체 및 보조제의 성분은, 알려진되었는지 확인합니다. 그것은 스테로이드 호르몬 (내 연구실에서 수행한 작업의 큰 부분은 스테로이드 호르몬의 행동을 조사 것을 주어) 피하기 위해 혈청 무료 / 숯불 송두리째 / 페놀 레드 무료로 매체를 사용하여 내 연구실에서 표준 방법입니다. 이 개념은 - 당신이 사용하고있는 솔루션이 뭔지 알아요 - 셀 culturing의 모든 단계에 중요합니다.

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Acknowledgments

JLN는 MH 68347에 의해 지원되었다.

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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