Préparation et entretien des neurones ganglions de la racine dorsale dans les cultures à compartiments

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Summary

Nous décrivons ici la technique de la préparation et l'entretien des chambres compartimentées pour les neurones sensoriels en culture des ganglions de la racine dorsale.

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

Neurones étendre les processus axonal qui sont loin du corps cellulaire pour innerver les tissus cibles, où la cible des facteurs de croissance dérivés sont nécessaires pour la survie des neurones et la fonction. Les neurotrophines sont spécifiquement requis pour maintenir la survie et la différenciation des neurones sensoriels innervant mais la question de savoir comment ces neurotrophines cibles dérivées de communiquer vers le corps cellulaire des neurones innervant a été un domaine de recherche actif depuis plus de 30 ans. Le modèle le plus communément acceptée de la façon dont les signaux atteignent neurotrophine du corps cellulaire propose que la signalisation endosomes réaliser ce signal rétrograde le long de l'axone. Afin d'étudier le transport rétrograde, un système de culture a été initialement conçu par Robert Campenot, dans laquelle les corps cellulaires sont isolés de leurs axones. La technique de préparation de ces chambres compartimentées pour les récapitule en culture des neurones sensoriels de la stimulation sélective des terminaisons neuronales qui se produit dans le communiqué suivant in vivo de cibles dérivées de neurotrophines. Rétrograde événements de signalisation qui exigent longue portée transport rétrograde microtubules dépendants ont des implications importantes pour le traitement des troubles neurodégénératifs.

Protocol

Préparation des réactifs

  1. Revêtement collagène: le collagène manteau de p35 plaques de culture tissulaire et les placer dans une étuve à 37 ° C pendant 2 jours avant le graissage des diviseurs. La concentration finale de collagène devrait être à 0,71 mg / ml dilué dans 0,001 N HCl. Puis, ajoutez 1 ml de mélange par plaque.
  2. Chargeurs de graisse: Afin de combler le chargeur de graisse, une seringue de 60 ml doit d'abord être rempli de graisse à vide Corning. Utiliser la seringue pour remplir le chargeur de la graisse, l'envelopper dans du papier et ensuite l'autoclave pendant 45 minutes.
  3. Diviseurs Téflon: les diviseurs peuvent être réutilisés après chaque expérience, mais doit d'abord être bien nettoyés. Retirez le diviseur de la plaque, essuyez toutes les graisses restantes et les placer dans l'acide sulfurique pendant 2 jours. Après avoir retiré de l'acide, rincer à l'eau 3X, faire bouillir pendant 20 minutes, laisser sécher, placer dans un plat en verre P100 de Pétri et l'autoclave pendant 20 minutes.
  4. N2-méthylcellulose: Peser 1,5 g de méthylcellulose et le placer dans une bouteille de 500 ml. Ajouter un barreau et il autoclave pendant 20 minutes à sec (à partir de ce point de tous les travaux doivent être stériles). Ensuite, ajoutez 500 ml de sérum médias libres (N 2), et mélanger dans une chambre froide jusqu'à ce qu'elle se dissolve. Aliquoter en 50 ml Coniques et congeler à -20 ° C. Pour le stock, partie aliquote de travail l'un des 50 Coniques mL dans des tubes de 1 ml et congeler à -20 ° C.
  5. DRG médias: DMEM, 5% de sérum de cheval inactivé par la chaleur, la streptomycine et 1% de pénicilline.
  6. 100ng/mL DRGN médias: La concentration des stocks de ces deux facteurs de croissance nerveuse (NGF) et le facteur neurotrophique dérivé du cerveau (BDNF) est 1mg/mL. Diluer chacun des neurotrophines 1:10.000 dans les médias DRG. Les cultures peuvent être cultivées dans NGF seul; cela modifie le complément de neurones qui survivent dans les cultures.

    Remarque: Si nécessaire, la concentration de (cytosine arabinoside) Arac est 1um et utilisé à une concentration finale de 0.3uM. Ce sera d'inhiber la croissance des cellules de Schwann et des cellules gliales d'autres.

  7. 10ng/mL DRGN médias: Diluer le 100ng/mL DRGN médias (01h10) avec les médias DRG.

    Figure 1


    Outils Figure 1. Nécessaire pour mettre en place

Mise en place des chambres compartimentées (démarrer ce processus 1-2 jours avant la dissection)

  1. Faire une égratignure au milieu d'un revêtues de collagène p35 plat avec un mouvement vers l'extérieur.
  2. 30ul Lieu de N2-méthylcellulose sur le milieu de la rayure. Set plats de côté jusqu'à ce diviseur est graissé.
  3. Fixez un adaptateur de calibre 23 talon de Luer pour le chargeur de graisse. Saisir le diviseur de Teflon avec une paire de pinces hémostatiques 90 ° d'angle et le poser à plat avec le diviseur vers le haut sous un microscope. Trace le diviseur avec de la graisse en s'assurant que chaque fois que l'adaptateur est placé à un nouveau point de départ de l'adaptateur est inséré dans la graisse de l'étape précédente afin qu'il y ait une ligne continue de la graisse (voir schéma). Une fois que la graisse est appliquée à l'ensemble du diviseur, tournez l'un des plats préparés p35 à l'envers et la placer de sorte que le N2-méthylcellulose est sur le compartiment du milieu. Appuyez sur le fond du plat avec une paire de pincettes. Veillez à appuyer sur l'intérieur de la cloison dans les quatre coins (en haut à gauche, en bas à droite, en haut à droite, en bas à gauche, indiqué dans le schéma par «X»).

    Figure 2

    Figure 2: Étapes pour graisser le diviseur

    Remarque: Il est important d'appuyer assez fermement pour que la graisse fait une étanchéité totale avec le plat, mais si trop de pression est ajoutée, les axones ne franchira pas dans les compartiments latéraux.

    Ramassez les pinces hémostatiques, retournez-le et débrider le diviseur. Enfin, placez le plat avec le diviseur fermement attaché sous le microscope se concentrant sur le fond du compartiment du milieu. Avec le chargeur de la graisse, faire une petite barrière (0,25 cm) de sorte qu'une fois que les cellules sont placées dans le compartiment central, ils ne peuvent pas s'échapper.
  4. Après avoir mis en place plusieurs cultures, les médias DRG place dans chacun des compartiments latéraux et les placer dans un incubateur dans lequel les cellules seront maintenues. Autoriser les cultures de s'asseoir pendant plusieurs heures et ensuite vérifier les fuites. Si les médias ont fui dans le compartiment du milieu, puis la culture est inutilisable.

    Remarque: Lors de la première formation à cette technique, il est important de mettre en place des cultures plus que nécessaire pour une expérience, que plusieurs seront étanches.

    Figure 3

    Figure 3: "Bonne vs fuient la« culture

Le maintien des neurones DRG dans les cultures compartimentées

  1. Jour 1: Remplacer DRG des médias dans les compartiments latéraux avec 100ng/mL DRGN médias + AraC. Effectuer la dissection et ajouter des cellules à compartiment central (100 000 cellules).
  2. Jour 2: Ajouter 10ng/mL médias + AraC à l'extérieur du diviseur de téflon jusqu'à ce que le flux média au cours de la barrière de la graisse fluide et les échanges avec le compartiment central.
  3. Jour 3: Remplacer les médias dans les compartiments latéraux pour 100ng/mL DRGN omettant l'ARAC et l'entourer de 10ng/mL DRGN omettant l'ARAC.
  4. Jour 6: Remplacer les médias dans les compartiments latéraux pour 1ng/ml + AraC et les entourer avec les médias DRG + AraC.
  5. Jour 9: Utilisation de l'expérimentation.


    Figure 4

    Figure 4: les images IHC des corps cellulaires et les axones distaux



    Remarque: Lorsque vous changez les médias, il est important d'aspirer le liquide de la partie supérieure du compartiment côté chacun. Aussi, ne jamais changer le support du compartiment milieu lui-même, seulement de l'entourer, et laissez couler sur la barrière de graisse dans le centre.

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Discussion

Dans cette vidéo, nous avons montré comment préparer et entretenir les chambres compartimentées pour une utilisation dans les neurones DRG culture. Fait correctement, ce système permet la séparation des corps de la cellule de l'axone, afin d'étudier les mécanismes par lesquels les neurotrophines signal à travers longs axones. Comme il est l'isolement fluidique entre les compartiments, il permet une stimulation sélective ou le traitement d'un compartiment, sans les autres compartiments touchés. Cultures de chambre à compartiments peuvent soutenir d'autres types cellulaires, y compris des neurones sympathiques du ganglion cervical supérieur, les neurones ganglionnaires de la rétine, et les neurones corticaux. Compréhension spatiale de la transduction du signal neurotrophine peut fournir des indications roman en traitements des maladies neurodégénératives. Plusieurs maladies neurodégénératives, y compris la maladie d'Alzheimer, maladie de Huntington et la maladie des motoneurones, sont associées à des anomalies dans le transport axonal. Des études récentes ont utilisé des chambres microfluidiques au lieu de ces chambres compartimentées. Les chambres microfluidiques 4,5 présentent plusieurs avantages pour l'analyse de l'imagerie.

Des études antérieures ont testé la capacité de ces cultures pour empêcher la diffusion entre l'axone et le compartiment corps cellulaire 1,3,6. Cela peut facilement être testée en ajoutant de faibles concentrations d'un colorant tel que le bleu trypan à un seul compartiment, et regarder pour la diffusion du colorant. Il devrait y avoir peu ou pas de diffusion visibles dans les 24 heures.

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Acknowledgements

Nous tenons à remercier Katharina Cosker et Stephanie Courchesne pour les discussions utiles.

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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