Preparazione e manutenzione dei neuroni dei gangli dorsali in Culture Compartmented

Published 10/17/2008
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Summary

Qui si descrive la tecnica di preparazione e mantenimento di camere compartimenti per la coltura dei neuroni sensoriali dei gangli delle radici dorsali.

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

I neuroni estendere i processi assoni che sono molto lontane dal corpo cellulare a innervare tessuti bersaglio, dove sono richiesti obiettivi derivati ​​fattori di crescita per la sopravvivenza neuronale e la funzione. Neurotrofine sono specificamente tenuti a mantenere la sopravvivenza e la differenziazione dei neuroni sensoriali che innervano ma il problema di come questi target-derivati ​​neurotrofine comunicare al corpo cellulare dei neuroni che innervano è stata un'area di ricerca attiva da oltre 30 anni. Il modello più comunemente accettata di come i segnali neurotrofina raggiungere il corpo cellulare propone di segnalazione endosomi portare questo segnale retrogrado lungo l'assone. Al fine di studiare il trasporto retrogrado, un sistema di coltura è stato originariamente ideato da Robert Campenot, in cui corpi cellulari sono isolati dal loro assoni. La tecnica di preparazione di queste camere compartimenti per la coltura dei neuroni sensoriali ricapitola la stimolazione selettiva di terminazioni neuronali che si verifica in vivo seguente comunicato del target di derivazione neurotrofine. Retrograda di segnalazione eventi che richiedono lunga distanza retrograda dipendente microtubuli hanno importanti implicazioni per il trattamento di patologie neurodegenerative.

Protocol

Preparazione dei reagenti

  1. Rivestimento collagene: collagene cappotto p35 piastre di coltura dei tessuti e mettere in forno a 37 ° C per 2 giorni prima ingrassaggio i divisori. La concentrazione finale di collagene deve essere di 0,71 mg / ml diluito in 0,001 N HCl. Poi, aggiungere 1 ml di miscela per piastra.
  2. Caricatori Grasso: Per riempire il caricatore grasso, una siringa da 60 ml deve prima essere riempita con grasso per vuoto Corning. Usare la siringa per riempire il caricatore grasso, avvolgetela in un foglio e poi in autoclave per 45 minuti.
  3. Divisori teflon: i divisori possono essere riutilizzati dopo ogni esperimento, ma deve prima essere puliti. Rimuovere il divisore dalla piastra, togliete tutti il ​​grasso residuo e il luogo in acido solforico per 2 giorni. Dopo aver rimosso dal lavaggio, sciacquare con acqua 3X, far bollire per 20 minuti, lasciare asciugare, mettere in un piatto di vetro P100 Petri e autoclave per 20 minuti.
  4. N2-metilcellulosa: Pesare 1,5 g di metilcellulosa e mettetelo in una bottiglia da 500 ml. Aggiungi un ancoretta e autoclave per 20 minuti a secco (da questo punto tutto il lavoro deve essere sterile). Successivamente, aggiungere 500 ml di siero multimediale gratuito (N 2), e mescolate in una stanza fredda fino a quando non si scioglie. Aliquota in 50 conicals ml e congelare a -20 ° C. Per lavorare stock, aliquota uno dei 50 conicals mL in provette 1 ml e congelare a -20 ° C.
  5. DRG media: DMEM, 5% di calore siero di cavallo inattivato, streptomicina e 1% di penicillina.
  6. 100ng/mL DRGN dei media: la concentrazione stock di entrambi fattore di crescita nervosa (NGF) e del cervello-derivato fattore neurotrofico (BDNF) è 1mg/mL. Diluire ciascuna delle neurotrofine 1:10.000 nei media DRG. Culture possono essere coltivate in NGF solo: questo altera il complemento di neuroni che sopravvivono nelle culture.

    Nota: Quando necessario, la concentrazione di (citosina arabinoside) AraC è 1um e utilizzato ad una concentrazione finale di 0.3uM. Questo inibire la crescita di cellule di Schwann e glia altri.

  7. 10ng/ml DRGN media: Diluire il 100ng/mL DRGN mezzi di comunicazione (1:10) con i media DRG.

    Figura 1


    Strumenti di Figura 1. Necessarie per set-up

La creazione di camere di compartimenti (avviare questo processo 1-2 giorni prima della dissezione)

  1. Fai un graffio nel bel mezzo di un piatto rivestito di collagene p35 con un movimento verso l'esterno.
  2. 30ul posto di N2-metilcellulosa al centro del graffio. Set piatti da parte fino divisore è ingrassato.
  3. Allegare una 23-gauge adattatore luer stub al caricatore grasso. Afferrare il divisore in teflon con un paio di hemostats angolo di 90 ° e adagiarlo orizzontalmente con il divisore rivolta verso l'alto sotto un microscopio. Traccia il divisore con grasso facendo in modo che ogni volta che l'adattatore è posto a un nuovo punto di partenza è inserito l'adattatore nella grasso dal passo precedente in modo che ci sia una linea continua di grasso (vedi schema). Una volta che il grasso viene applicato il divisore intero, girare uno dei piatti preparati p35 a testa in giù e posizionarlo in modo che il N2-metilcellulosa si trova sopra il vano centrale. Premere sul fondo del piatto con un paio di pinzette. Assicurarsi di premere sulla parte interna del divisore in quattro angoli (in alto a sinistra, in basso a destra, in alto a destra, in basso a sinistra, indicato nello schema da "X").

    Figura 2

    Figura 2: Procedura per ingrassare il divisore

    Nota: è importante per premere con forza sufficiente in modo che il grasso fa un sigillo completo con il piatto, ma se troppa pressione si aggiunge, gli assoni non attraverserà negli scompartimenti laterali.

    Sollevare il hemostats, capovolgerlo e sbloccare il divisore. Infine, posto il piatto con il divisore saldamente sotto il microscopio di messa a fuoco sul fondo del vano centrale. Con il caricatore grasso, fare una piccola barriera (0,25 centimetri) in modo che una volta che le cellule si trovano nel vano centrale non possono fuoriuscire.
  4. Dopo aver creato diverse culture, media luogo DRG in ciascuno dei compartimenti laterali e posto in una incubatrice in cui le cellule saranno mantenute. Lasciare le culture di sedersi per diverse ore e poi verificare la presenza di perdite. Se il supporto è trapelato nel vano centrale, quindi la cultura è inutilizzabile.

    Nota: La prima volta che questa tecnica di apprendimento, è importante impostare le culture più di quelli necessari per un esperimento, come molti si perde.

    Figura 3

    Figura 3: la cultura "Good vs leaky"

Mantenere i neuroni DRG nelle culture compartimenti

  1. Giorno 1: Sostituire DRG media negli scomparti laterali con 100ng/mL DRGN supporti + AraC. Eseguire la dissezione e aggiungere le celle a scomparto centrale (100.000 cellule).
  2. Giorno 2: Aggiungi 10ng/ml media + AraC verso l'esterno del divisore Teflon fino a quando il flusso dei media oltre la barriera grasso e liquidi gli scambi con il vano centrale.
  3. Giorno 3: Sostituire i media nei compartimenti laterali per 100ng/mL DRGN omettendo il Arac e il surround con 10ng/ml DRGN omettendo il AraC.
  4. Giorno 6: sostituire i media nei compartimenti laterali per 1ng/mL + Arac e il surround con DRG supporti + AraC.
  5. Giorno 9: Usa per la sperimentazione.


    Figura 4

    Figura 4: immagini IHC di corpi cellulari e gli assoni distali



    Nota: Quando si cambiano i media, è importante per aspirare il liquido dalla parte superiore di ogni singolo vano laterale. Inoltre, non cambia il contenuto del vano centrale stessa, solo dal surround, e lasciate fluire oltre la barriera del grasso verso il centro.

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Discussion

In questo video, abbiamo dimostrato come preparare e mantenere camere compartimenti per l'uso nei neuroni DRG coltura. Fatto correttamente, questo sistema consente la separazione del corpo cellulare dal assone al fine di studiare meccanismi attraverso i quali neurotrofine segnale attraverso lunghi assoni. Poiché non vi è l'isolamento fluidico tra i compartimenti, permette per la stimolazione selettiva o il trattamento di un compartimento senza altri compartimenti essere colpiti. Culture camera compartimentate in grado di supportare altri tipi cellulari tra cui i neuroni simpatici dai gangli cervicale superiore, i neuroni gangliari della retina, e neuroni corticali. Comprensione spaziale di trasduzione del segnale neurotrofina possono fornire indicazioni romanzo in trattamenti di malattie neurodegenerative. Diverse malattie neurodegenerative, tra cui il morbo di Alzheimer, morbo di Huntington e la malattia dei motoneuroni, sono associate a difetti nel trasporto assonale. Recenti studi hanno usato le camere microfluidica al posto di queste camere compartimentati. Le camere di microfluidica 4,5 hanno diversi vantaggi per l'analisi di imaging.

Precedenti studi hanno testato la capacità di queste culture per prevenire la diffusione tra l'assone e il vano corpo cellulare 1,3,6. Questo può essere facilmente testato con l'aggiunta di basse concentrazioni di un colorante come il blu tripano ad un solo vano, e cercare la diffusione del colorante. Ci dovrebbe essere poca o nessuna diffusione visibile entro 24 ore.

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Acknowledgements

Vorremmo ringraziare Katharina Cosker e Stephanie Courchesne per utili discussioni.

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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