Подготовка и обслуживание Спинной корневой Ganglia Нейроны в отсеки культур

Published 10/17/2008
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Summary

Здесь мы опишем технику подготовки и поддержания отсеки камеры для культивирования сенсорных нейронов спинного ганглиев корня.

Cite this Article

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F. Pazyra-Murphy, M., A. Segal, R. Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures. J. Vis. Exp. (20), e951, doi:10.3791/951 (2008).

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Abstract

Нейроны расширить аксонального процессы, которые далеки от тела клетки к иннервируют тканях-мишенях, где целевая полученных факторов роста, необходимых для выживания нейронов и функции. Нейротрофинов специально обязаны поддерживать выживание и дифференциации иннервирующих сенсорных нейронов, но вопрос о том, как эти целевые полученных нейротрофинов сообщает тела клетки нейронов, иннервирующих была областью активных исследований на протяжении более 30 лет. Наиболее общепринятой моделью того, как нейротрофина сигналы достигают тела клетки предлагает сигнализации эндосомы нести этот сигнал ретроградно вдоль аксона. С целью изучения ретроградного транспорта, культуры система первоначально была разработана Робертом Campenot, в которых клетка тела изолированы от своих аксонов. Техника подготовки этих отсеки камеры для культивирования сенсорных нейронов повторяет селективной стимуляции нейронов терминалов, что происходит в естественных условиях после выпуска целевых полученных нейротрофинов. Ретроградная сигнализация событий, требующих дальнего микротрубочек зависящих транспортных ретроградной иметь важные последствия для лечения нейродегенеративных расстройств.

Protocol

Подготовка реагентов

  1. Коллаген покрытие: коллаген пальто p35 культуре ткани пластин и поместить в духовку при температуре 37 ° С в течение 2 дней до смазки перегородок. Конечная концентрация коллагена должны быть в 0,71 мг / мл разведенного в 0,001 N HCl. Затем добавьте 1 мл смеси на чашку.
  2. Смазка погрузчики: Для того, чтобы заполнить смазкой погрузчика, 60 мл шприца сначала должен быть заполнены смазкой вакуумных Corning. Используйте шприц для заполнения смазкой погрузчик, заверните его в фольгу, а затем автоклаве в течение 45 минут.
  3. Тефлон делителей: Делители могут быть использованы повторно после каждого эксперимента, но сначала должен быть надлежащим образом очищены. Удалить делителя с пластинки, вытрите все оставшиеся жир и место в серной кислоте в течение 2 дней. Сняв с кислотой, промыть водой 3X, кипятить 20 минут, дать высохнуть, поместите в стеклянную p100 Петри и автоклаве в течение 20 минут.
  4. N2-метилцеллюлозы: отвесить 1,5 г метилцеллюлозы и поместить его в 500 мл бутылки. Добавить мешалкой и автоклава в течение 20 минут на сухой (с этой точки все работы должны быть стерильными). Затем добавьте 500 мл сыворотки свободных средств массовой информации (N 2), и движение в холодной комнате, пока он не растворится. Алиготе в 50 мл conicals и заморозить при -20 ° C. Для работы акции, аликвоту одного из 50 мл в 1 мл conicals трубы и замерзать при -20 ° C.
  5. DRG СМИ: DMEM, 5% тепла инактивированной лошадиной сыворотки и 1% пенициллина стрептомицин.
  6. 100ng/mL DRGN СМИ: фондовый концентрации как фактор роста нервов (ФРН) и мозгового нейротрофического фактора (BDNF) является 1mg/mL. Развести каждый из нейротрофинов 1:10000 в DRG СМИ. Культуры могут быть выращены в NGF в одиночку, это меняет дополнение нейронов, которые выживают в культуре.

    Примечание: При необходимости концентрации (цитозинарабинозид) AraC является 1 мкм и использоваться при конечной концентрации 0.3uM. Это будет тормозить рост клеток Шванн и другие глии.

  7. 10ng/mL DRGN СМИ: Развести 100ng/mL DRGN СМИ (1:10) со средствами массовой информации DRG.

    Рисунок 1


    Рисунок 1. Инструменты, необходимые для ввода в эксплуатацию

Настройка отсеки камер (начать этот процесс за 1-2 дня до вскрытия)

  1. Сделать нуля в середине коллагена покрытием p35 блюдо с внешним движением.
  2. Место 30ul из N2-метилцеллюлозы на середине нуля. Установить блюда в сторону, пока делителя смазаны.
  3. Прикрепить 23 калибра Luer адаптер кабеля, идущего к смазкой погрузчика. Возьмитесь за делителя тефлон с парой углом 90 ° hemostats и положите его плашмя с делителя вверх под микроскопом. Трассировка делителя смазкой убедившись, что каждый раз, когда адаптер устанавливается на новую точку отсчета Адаптер вставляется в жир из предыдущего шага, так что существует непрерывная линия смазкой (см. схему). Как только смазка применяется ко всему делителя, включите одну из подготовленных p35 блюда с ног на голову и поместите его так N2-метилцеллюлозы составляет более среднего отсека. Нажмите на дно тарелки с пинцетом. Убедитесь, что нажали на внутренней стороне делителя в четырех углах (верхняя левая, нижняя правая, верхняя правая, нижняя левая, указанным в диаграмме "X").

    Рисунок 2

    Рисунок 2: Шаги для смазывания делителя

    Примечание: Очень важно, чтобы пресса достаточно крепко, так что жир делает полную герметизацию с блюдо, но если слишком много давления добавил, аксоны не будет пересекать в боковые отсеки.

    Возьмите hemostats, перевернуть его и разжимать делителя. И наконец, место блюдо с делителя прочно прикреплены под микроскопом акцентом на нижней части среднего отсека. С жиром погрузчик, сделать небольшой барьер (0,25 см), так что, как только клетки помещаются в среднем отсеке, они не могут просочиться наружу.
  4. После того, как создали несколько культур, место DRG СМИ в каждой из бортовых отсеков и место в инкубаторе, в котором клетки будут сохранены. Разрешить культур, чтобы сидеть в течение нескольких часов, а затем проверить на наличие утечек. Если средства массовой информации просочились в среднем отсеке, то культура является непригодным для использования.

    Примечание: При первом обучения этой технике, очень важно создать более культур, чем нужно для эксперимента, так как ряд будет вытекающих.

    Рисунок 3

    Рисунок 3: «Хорошие результаты против вытекающей" культура

Поддержание DRG нейроны в отсеки культур

  1. День 1: Замените DRG средств массовой информации в бортовых отсеков с 100ng/mL DRGN СМИ + AraC. Выполнить вскрытие и добавления ячеек в центр отсека (100 000 клеток).
  2. День 2: Добавить 10ng/mL СМИ + AraC для внешнего делителя тефлон, пока СМИ потоков через барьер жира и обмена жидкости с центром отсека.
  3. День 3: Замените средств массовой информации в бортовых отсеков для 100ng/mL DRGN опустив AraC и окружить 10ng/mL DRGN опустив AraC.
  4. День 6: Замените средств массовой информации в бортовых отсеков для 1ng/mL + AraC и окружить DRG СМИ + AraC.
  5. День 9: Используйте для экспериментов.


    Рисунок 4

    Рисунок 4: IHC изображения клеточных тел и дистальных аксонов



    Примечание: При изменении медиа, важно для аспирации жидкости из верхней части каждой стороны отсека. Кроме того, никогда не меняются СМИ с середины отсека, а только от окружающих, и пусть он обтекании смазки барьер в центре.

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Discussion

В этом видео, мы показали, как готовить и поддерживать отсеки камеры для использования в нейронах DRG культивирования. Было сделано правильно, эта система позволяет разделение тела клетки от аксона с целью изучения механизмов, посредством которых нейротрофинов сигнала на большие аксонов. Так как жидкостный изоляцию между отсеками, оно позволяет для селективной стимуляции или лечение одного отсека без остальных отсеков вреда для себя. Культур на отсеки, камера может поддерживать другие типы клеток, в том числе симпатических нейронов из верхних шейных ганглиев, нейронов сетчатки ганглия, и корковые нейроны. Пространственное понимание передачи сигнала нейротрофина может предоставить новые идеи в лечении нейродегенеративных расстройств. Несколько нейродегенеративных заболеваний, включая болезнь Альцгеймера, болезнь Хантингтона и заболевания двигательных нейронов, которые связаны с дефектами в аксонального транспорта. Недавние исследования использовались микрожидкостных камерах вместо того, чтобы эти отсеки камер. Микрожидкостных камеры 4,5 имеют ряд преимуществ для работы с изображениями анализа.

Предыдущие исследования протестировали способность этих культур для предотвращения диффузии между аксонов и отсек ячейки тела 1,3,6. Это можно легко проверить, добавив низкие концентрации красителя, таких как трипанового синего до одного отсека только и ищите диффузии красителя. Там должно быть мало или нет видимых диффузии в течение 24 часов.

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Acknowledgements

Мы хотели бы поблагодарить Катарина Cosker и Стефани Courchesne за полезные обсуждения.

Materials

Name Type Company Catalog Number Comments
collagen Reagent BD Biosciences 354249
N2-methylcellulose 400CPS Reagent Sel-Win Chemicals
Teflon divider Other Tyler Research CAMP10 many other types of dividers are available
Pin rake Tool Tyler Research Camp-PR
Grease loader Tool Tyler Research Camp-GLSS
DMEM Reagent Fisher Scientific MT10017CV
NGF Reagent PeproTech Inc 450-01
BDNF Reagent PeproTech Inc 450-02
High vacuum grease Reagent Fisher Scientific 14-635-5D
AraC Reagent Sigma-Aldrich C-1768
23 gauge luer stub adapter Tool Fisher Scientific 427565
90° angle hemostats Tool Roboz Surgical Instruments Co. RS-7035

DOWNLOAD MATERIALS LIST

References

  1. Campenot, R. B. Independent Control of the Local Environment of Somas and Neurites. Methods in Enzymology. 58, 302-307 (1979).
  2. Watson, F. L., et al. Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nature Neuroscience. 4, 981-988 (2001).
  3. Heerssen, H. M., et al. Dynein motors transport activated Trks to promote survival of target-dependent neurons. Nature Neuroscience. 7, 596-603 (2004).
  4. Taylor, A. M., et al. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods. 2, 599-605 (2006).
  5. Park, J. W., et al. Microfluidic culture platform for neuroscience research. Nat Protoc. 4, 2128-2136 (2006).
  6. Ure, D. R., et al. Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures. J Neuroscience. 4, 1282-1290 (1997).

Comments

49 Comments

  1. Hi I have just watch your presentaion on the Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures and it was very impressive. I am intersted in the function of the methylcellulose, dŒs it provide a space through which the axons can pass, do the cell bodies settle on the collagen surface. Regards Paul

    Reply
    Posted by: Anonymous
    November 19, 2008 - 7:27 AM
  2. Hi Paul-
    Thank you for your comment.  The cell bodies do settle onto the collagen surface.  As for the small amount of N²-methylcellulose that is placed on the center of the scratch, the media acts as a space for the axons to extend easily on and the methylcellulose is used to thicken the media slightly. 

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    December 15, 2008 - 3:06 PM
  3. Hi, your method looks simple and powerfull. It's possible to make an immuno staining without to remove the teflon divider? DŒs the divider cause troubles during immunofluorescence staining for example? Regards Giuseppe 

    Reply
    Posted by: Anonymous
    December 13, 2008 - 7:35 AM
  4. Hi Giuseppe-
    Thank you for your comment. Fix, wash and add antibodies as usual within the compartments for immunostaining. Remove the divider, carefully wipe away excess grease and coverslip before imaging.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    December 15, 2008 - 3:52 PM
  5. Hi Maria,

    I had a question about immunostaining. So in reference to your above comment, you keep the teflon dividers in place when you fix and add antibodies, and only remove it at the end of immunostaining? Is anything else different?

    Thanks,
    Supraja.

    Reply
    Posted by: Anonymous
    October 22, 2010 - 7:01 PM
  6. Hi Supraja-
    Yes, only remove the divider at the end of the immunostaining. Everything else is done exactly the same.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    October 25, 2010 - 3:42 PM
  7. Hi, beautiful work, congratulations! Have you tried to cultivate adult DRG also? Best regards and good luck! Otilia

    Reply
    Posted by: Anonymous
    February 5, 2009 - 6:34 AM
  8. Hi Otilia- Thank you for your comment.  We have not tried cultivating adult DRGs in this system.   Good Luck, Maria

    Reply
    Posted by: Anonymous
    March 19, 2009 - 12:03 PM
  9. Dear Otilia,

    I too was really impressed by Maria's work and wanted to use the technique for my work. I just wanted to let you know that I have been using adult DRGs in this system and it still works brilliantly.

    Best regards,

    Philippa

    Reply
    Posted by: Anonymous
    September 2, 2009 - 1:02 PM
  10. Thanks a lot, Philippa!

    Reply
    Posted by: Anonymous
    January 18, 2010 - 4:08 PM
  11. Hi, I hvae not been able to watch the video.  Could you please email me  a copy. Sincerely, Supinder Bedi, Ph.D. University of Texas, Houston

    Reply
    Posted by: Anonymous
    February 11, 2009 - 3:39 PM
  12. Hi, We are very interested in your method, that’s great!
    We have one question: on day1, how do you "perform dissection and add cells to center compartment (100,000cells)"?  Can you please provide more details? Best regards Yi

    Reply
    Posted by: Anonymous
    March 5, 2009 - 3:52 PM
  13. Hi Amazing video; really impressive and well constructed. I'm hoping to use the compartmented culture system for some of my experiments and so am currently trying to set it up however as i hope to do immunostaining of the cultures I was just wondering whether you use just normal polystyrene cell culture dishes or glass-bottomed dishes/ dishes with a glass coverlip plated in it? If you use glass what thickness and type of glass do you use? The reason I ask is I've been seeing whether I can coat glass coverslips with collagen in order to be able to detach them from the dishes and mount them onto microscope slides later however I don't seem to be able to coat the borosilicate glass coverslips that I have. I've tried all sorts of methods including your protocol here but it seems that the collagen dŒsn't want to stick to the glass. They are definitely degreased throughly therefore I was wondering whether it was an issue with the charge of the glass and whether there was a different sort of glass I should be using. Any suggestions would be really appreciated. Best regards, Philippa

    Reply
    Posted by: Philippa M.
    March 19, 2009 - 7:25 AM
  14. Hi Philippa- Thank you for your comment.  We only use polystyrene cell culture dishes that have been coated with collagen.  It is impossible to make the necessary scratches on the glass.  We often do immunostaining and image with this system, however, there is another system, microfluidic chambers, that may give you higher quality images. Good Luck, Maria  

    Reply
    Posted by: Anonymous
    March 19, 2009 - 11:56 AM
  15. Dear Philippa, I have seen the video and read your comment only yesterday, but maybe this can help: at www.mattek.com, you can order 35mm or 50mm dishes with a partially glass bottom (coverslip that you can even take out afterwards and that is either uncoated or coated with collagen or poly-D-lysine). We have been using them on a regular basis for live cell imaging and they're very useful. From my side, I was wondering if you made any progress with scratching glass dishes, because I would like to do this for my particular experiment. Best regards, Katrien

    Reply
    Posted by: Katrien J.
    August 28, 2009 - 5:01 AM
  16. Hi Katrien,

    Thank you for your suggestions. In answer to your question unfortunately I wasn't able to scratch the glass coverslips. Unfortunately I think it would be a case of having to get them engraved independently. I've been using the polystyrene cell culture dishes since and although maybe the optics aren't as good as they could I'm actually still able to obtain very good images after immunostaining with the inverted microscope. So if this is similar to what you plan to do it might be worth a try. Best of luck.

    Philippa

    Reply
    Posted by: Anonymous
    September 2, 2009 - 3:29 AM
  17. Hi Philippa, thanks for your answer. Would it be possible to let me know what magnification objective you use and what kind of structures you are looking at, just to have an idea if this might work for me as well?
    Thank you and best regards, katrien

    Reply
    Posted by: Katrien J.
    September 2, 2009 - 3:38 AM
  18. Of course: I work with a ²0x magnification and take a series of consecutive images of the neurites that have extended into the side compartments in order to reconstruct the whole image and assess neurite outgrowth before and after a treatment. I would prefer to use 10x magnification but we don't have the lens for it. Hope that helps. Philippa

    Reply
    Posted by: Anonymous
    September 2, 2009 - 12:59 PM
  19. Hi Amazing video; really impressive and well constructed. I'm hoping to use the compartmented culture system for some of my experiments and so am currently trying to set it up however as i hope to do immunostaining of the cultures I was just wondering whether you use just normal polystyrene cell culture dishes or glass-bottomed dishes/ dishes with a glass coverlip plated in it? If you use glass what thickness and type of glass do you use? The reason I ask is I've been seeing whether I can coat glass coverslips with collagen in order to be able to detach them from the dishes and mount them onto microscope slides later however I don't seem to be able to coat the borosilicate glass coverslips that I have. I've tried all sorts of methods including your protocol here but it seems that the collagen dŒsn't want to stick to the glass. They are definitely degreased throughly therefore I was wondering whether it was an issue with the charge of the glass and whether there was a different sort of glass I should be using. Any suggestions would be really appreciated. Best regards, Philippa

    Reply
    Posted by: Philippa M.
    March 19, 2009 - 8:12 AM
  20. Hi Amazing video; really impressive and well constructed. I'm hoping to use the compartmented culture system for some of my experiments and so am currently trying to set it up however as i hope to do immunostaining of the cultures I was just wondering whether you use just normal polystyrene cell culture dishes or glass-bottomed dishes/ dishes with a glass coverlip plated in it? If you use glass what thickness and type of glass do you use? The reason I ask is I've been seeing whether I can coat glass coverslips with collagen in order to be able to detach them from the dishes and mount them onto microscope slides later however I don't seem to be able to coat the borosilicate glass coverslips that I have. I've tried all sorts of methods including your protocol here but it seems that the collagen dŒsn't want to stick to the glass. They are definitely degreased throughly therefore I was wondering whether it was an issue with the charge of the glass and whether there was a different sort of glass I should be using. Any suggestions would be really appreciated. Best regards, Philippa

    Reply
    Posted by: Philippa M.
    March 19, 2009 - 2:04 PM
  21. Hi,   Could you please elaborate a bit more on the DRG dissection and how you get to 100,000 cells please Thanks,   Gustavo Ayala R. Clarence and Irene H Fullbright Chair in Pathology Professor Baylor College of Medcine  

    Reply
    Posted by: Anonymous
    March 24, 2009 - 4:58 PM
  22. hi, thank you for the presentation, your novel model for preparation of drg neurons seem to be very efficient. can you please elaborate how did you get the neurons and how old was the rat fetus? thanks ahead, Amit Moran, moranamit@gmail.com    

    Reply
    Posted by: Anonymous
    March 30, 2009 - 11:58 PM
  23. It is very interesting. I am wondering regarding the use of NGF. You have used Recombinant human beta NGF. Is there any special reason you used human NGF? or is it ok that we can use rat NGF?

    Reply
    Posted by: Anonymous
    June 23, 2009 - 3:16 PM
  24. Hi Anand-

    We prefer to use the recombinant human NGF but it is certainly okay to use rat NGF.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    June 30, 2009 - 12:51 PM
  25. Thanks for your presentation. Have you ever tried cultivating hippocampal neurons? If it 's, what did you coated with your dish, collagen or poly-l-lysine?

    Regards


    Mei

    Reply
    Posted by: Anonymous
    August 18, 2009 - 4:35 PM
  26. Hi Mei-

    Sorry, we have never cultivated hippocampal neurons using this system. There is a paper, Ivins et al., 1998, that uses a modified compartmented culture system that you may find helpful.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    August 21, 2009 - 1:39 PM
  27. Dear colleges, thank you very much for nice performance and demonstration of this method!
    I have a question. How do you think could I use this compartmented culture to investigated axonal degeneration. If I will add a substance under the investigation to axonal part of chamber, how could I be sure that this compound dŒs not penetrate to cell body part?
    Thank you very much for answer beforehand and good luck in your future experiment!!!

    Reply
    Posted by: Liudmila E.
    September 28, 2009 - 9:17 AM
  28. Hi Lula-
    If set up properly (no leakage) these chambers are fluidically isolated.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:54 PM
  29. Ok, how it could be isolated?: side parts and middle parts fluidically, if even neurites can pass through this barrier. That means that compounds (chemical substance) can do it also. am I wrong?

    Reply
    Posted by: Anonymous
    January 14, 2010 - 4:49 AM
  30. Hi Maria,
    I will like to confirm the concentration of collagen coating that you use have on the protocol (0,71mg/mL diluted in 0,001N HCl). In our lab we use collagen coating for cultures and the concentration is very less.
    Regards
    AleMorán

    Reply
    Posted by: Anonymous
    September 28, 2009 - 1:09 PM
  31. Hi Alemor-
    Yes, that is the correct concentration of collagen.
    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:39 PM
  32. Thanks Maria,
    I would like to ask you a new cuestion. Did you cut de needle to avoid de sharp point?
    Regards

    Reply
    Posted by: Anonymous
    March 30, 2010 - 11:29 AM
  33. Hi-
    The adapter that we use has a blunt end. It's a ²3 gauge luer stub adapter (Fisher cat #4²7565).
    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    March 30, 2010 - 2:55 PM
  34. Hi, It is quite interesting. I need some help in isolating DRG. I tried. But not able to identify them. Can you pls help me.
    Thanks in advance.

    Reply
    Posted by: Anonymous
    October 26, 2009 - 3:44 PM
  35. Hi Anand-
    The DRGs are located along each side of the spinal cord. In the E15 rats, the DRGs are clearly visible as ganglia along the spinal cord. At older ages the DRGs are encased within the vertebrae.
    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:42 PM
  36. Hi,
    Great presentation!
    I was wondering, what antibodies did you use for the IHC images?

    Thanks,
    Amy

    Reply
    Posted by: Amy M.
    December 1, 2009 - 8:43 PM
  37. Hi,

    many thanks for posting this really helpful video. When you apply the grease on the divider do you work under a hood? If not, how do you maintain sterility? I have tried working outside a hood and I have had problem of contamination (all solutions, and tools have been either filtered or autoclaved)

    Kind regards,

    Ale

    Reply
    Posted by: Anonymous
    January 13, 2010 - 11:31 AM
  38. Hi Ale-
    It would be best if you set up the cultures in a dissecting hood if you are having problems with contamination. We use UV to sterilize the area but the hood that we work under has no air flow and is therefore not a completely sterile environment.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:51 PM
  39. Hi
    Beautiful work! And I have been tried to constructed the compartmented cultures for many times but failed .I don't know why. I wonder how do you get to 100,000 cells .Could you please elaborate a bit more on the DRG dissection .In my compartmented cultures ,there are only a few axons across the barrier and get to the distal compartment.I have tried it for many times but the results are the same.I guss if the cell differentiation is not good enough or something wrong with the applying grease.I have no idea .So i think if you can give me your email and I have many problems to ask you.My email is jjdongch@gmail .com. I am looking forward to your reply .Thank you!

    Reply
    Posted by: jingjing d.
    May 17, 2012 - 7:39 AM
  40. Hi!, excellent video!. We are reproducing this system, but now, we have problem with collagen coating. It does not gelled 3 or 5 days after!, in fact, it does not change. We´re using the same collagen (BD Bioscience ,354249) and your final concentration (0.71mg/ml diluted in 0.001N HCl). What recommendations do you have?.

    Kind Regards!

    Reply
    Posted by: DIANA M.
    January 9, 2014 - 1:00 PM
  41. Hi Diana-
    The plates need to be put in a dry oven at 37 degrees for 3 days. Are you doing that? Also, the collagen doesn't gel, it dries completely. Good luck and feel free to email me with any additional questions. maria_pazyra@dfci.harvard.edu

    Reply
    Posted by: Anonymous
    January 9, 2014 - 1:29 PM
  42. Great article. What are the product numbers and companies for methylcellulose and N2 serum free media?

    Reply
    Posted by: Eric W.
    June 17, 2014 - 3:04 PM
  43. Hi Eric- The methylcellulose we currently use is from Xenex (catalog # E4M) and the serum free media is just plain DMEM. Good Luck, Maria

    Reply
    Posted by: Anonymous
    June 17, 2014 - 3:21 PM
  44. Hi, Maria
    Your article and video in jove helped our research a lot, excellent!
    I have one question. Are you still using N2-MC? Please let me know, if there is better one that can prevents grease from closing grooves.
    Thank you
    S
    Thank you

    Reply
    Posted by: Shingo K.
    October 13, 2015 - 11:31 AM
  45. Hi Shingo-
    I'm not sure I understand your question. Are your axons not growing through the grooves? The N2 is used to help the axons slide under the grease and we still use the Xenex brand. If your axons are not growing through it is probably more about how much pressure you are applying when you press the dish to the teflon divider.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    October 13, 2015 - 11:54 AM
  46. Hi Maria-
    Thanks for your quick reply.
    Axons do grow well into the next chamber with N2-MC, but not through all the grooves I made (~50%). I am wondering if there is other potential substance (i.e., one with higher viscosity) that I can try...
    I am expecting more... or do you think ~50% is OK ?
    shingo

    Reply
    Posted by: Shingo K.
    October 13, 2015 - 1:53 PM
  47. Shingo-
    My suggestion would be to add more N2 to cover more of the grooves. I don't know of any other substance that would work. 50% is actually pretty good.

    Best,
    Maria

    Reply
    Posted by: Anonymous
    October 13, 2015 - 2:09 PM
  48. Hi Maria,

    Thank you for publishing this protocol. I have been trying to grow cortical neurons in a custom made Teflon divider (1.5mm width of central compartment and 0.8mm height of divider), without much success. The cell bodies leak in the side compartments.
    I have coated the dish with poly-d-lysine instead of collagen do you think this can affect the chamber set up? Have you ever tried to culture cortical neurons in this chamber or know someone who has? I'm thinking that perhaps the cell body of cortical neurons is smaller than DRGs and this could cause leakage, what do you think?

    Regarding the methylcellulose step. I diluted it in Neurobasal (already supplemented with Pen Strep and Glutamax). I've added it on the middle of the scratched region and then applied the divider with grease in the same way as you explain in the video.I removed it before adding the cells but I did not let it dry. Are you supposed to leave the drop medium with methylcellulose when cells are added? How long do the cells need before they attach to the dish completely?

    Any advice on how to improve my method would be highly appreciated.

    Thanks a lot in advance,

    Anna

    Reply
    Posted by: University of Aberdeen .
    April 5, 2016 - 2:05 PM
  49. Hi Anna-
    We have never cultured cortical neurons in this system before but I do think that the collagen or matrigel (that's what we use now) is essential. I don't think the size of the cells is a factor. It's probably more about the grease. And mastering that only comes with lots and lots of practice. Are you using these for biochemistry? If you are using them for staining or imaging you should look into microfluidic chambers. We use those in the lab too. We leave the methylcellulose in the center compartment so no need to remove it before plating the cells....and the cells should attach within a couple of hours. Good luck and please feel free to email me with any further questions. maria_pazyra@dfci.harvard.edu

    Best,
    Maria

    Reply
    Posted by: Anonymous
    April 5, 2016 - 3:34 PM

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