Primäre Kultur des Hippocampus-Neuronen von neugeborenen Ratten P0

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Summary

Die Dissektion und das Wachstum der Zellen von einem einzelnen Bereich des Gehirns erleichtert Untersuchung zellulärer und physiologischer Parameter. Wir beschreiben ein Verfahren zur Primär-Zellkultur, die Neuron-angereicherten Kulturen produziert in einem Serum-freien Umgebung.

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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

Die physiologischen Eigenschaften von Neuronen im Hippocampus werden häufig untersucht, vor allem wegen der Beteiligung des Hippocampus beim Lernen und Erinnern. Primäre hippocampale Zellkultur ermöglicht Neurowissenschaftler die Aktivität und Eigenschaften von Neuronen in den einzelnen Zellen und einzelne Synapse-Ebene zu untersuchen. In diesem Video zeigen wir, wie zu isolieren und zu wachsen primäre Zellen des Hippocampus von neugeborenen Ratten. Der Hippocampus kann von jedem neugeborenen Tieren in so kurz wie 2-3 Minuten isoliert werden, und die Kulturen kann beibehalten werden bis zu zwei Wochen. Wir werden auch kurz zeigen, wie diese Neuronen im Hippocampus für ratiometrische Calcium-Imaging verwenden. Während dieses Protokoll beschreibt den Prozess für den Hippocampus, mit wenig bis gar keine Änderung, kann es auf andere Regionen des Gehirns eingesetzt werden.

Protocol

Vor Hippocampus Isolation

Vor Beginn der Hippocampus Isolation, um sicherzustellen, dass alle Werkzeuge, die steril sind. Spray auf der Kultur Haube mit 70% Ethanol, und legen Sie die Werkzeuge in der Motorhaube. Sie benötigen 6 - und 10-cm-Petrischalen, steril Poly-L beschichteten Deckgläsern, Pipetten und Spitzen, Einweg-Pipetten und einem elektrischen Pipette. Von diesem Punkt an, denken Sie daran, richtige sterile Technik zu verwenden.

  1. Schalten Sie das Wasserbad und stellen Sie sicher, dass es bis zu 37 ° C erhitzt
  2. Die folgenden Lösungen, die bei 4 ° C gelagert werden benötigt werden:
    • Modifiziertem Eagle-Medium (MEM)
    • Neurobasal
    • Hanks Salzlösung (HBSS)
    • Boratpufferlösung
    • Natriumpyruvat Lösung
    • Sterilfiltriert 20% Glucose-Lösung in destilliertem Wasser

    • Achten Sie darauf, alle Flaschen abspritzen mit 70% Ethanol, bevor Sie sie in der Motorhaube.

  3. Nehmen Sie diese gefrorenen Lösungen aus der Tiefkühltruhe und legen Sie sie in einem beheizten Wasserbad:
    • Ein 5 ml Aliquot Pferdeserum
    • Ein 1 ml Aliquot B-27 Ergänzung
    • Ein 1 ml Aliquot 100X Antibiotika (Penicillin und Streptomycin)
    • Und ein 0,5 ml Aliquot von L-Glutamin
  4. Nachdem die Lösungen aufgetaut haben, nehmen sie aus dem Wasserbad, sprühen Sie sie mit 70% Ethanol, und legen Sie sie in der Motorhaube. Nun ist die Beschichtung und Neurobasalmedium hergestellt werden kann.
  5. Nachdem die Lösungen bereit sind, verschließen Sie die Behälter dicht und legen Sie sie in den 37 ° C-Bad zum Aufwärmen während der Durchführung der Hippocampus Isolation.
  6. Auch wird ein aliquoter der Protease Trypsin (2,5%) aus der Tiefkühltruhe und legen Sie sie in das Wasserbad. Trypsin wird verdauen seziert Hippocampus, die im nächsten Schritt isoliert werden.
  7. Nehmen Sie einen 15 ml konische Röhre, füllt sie mit HBSS, und beschriften Sie sie mit der behandelten Gruppe. Es ist hier, dass isolierte hippocampi im nächsten Schritt werden gesammelt.

Hippocampus Isolation

  1. Zu Beginn des Hippocampus Isolation, stellen Sie sicher, neugeborenen Welpen sind sauber und haben ihre Milch Bands, Plazenta, Nabelschnur von ihrer Mutter entfernt.
  2. Platzieren Sie den Welpen in einer Petrischale, Spray mit 70% Ethanol zu reinigen, und legen Sie sie in der Motorhaube. Die Tiere werden unmittelbar vor der Kultivierung eingeschläfert.
  3. Für weitere Sterilisation nehmen einen Welpen aus der Schüssel, tauchen die Welpen in 70% Ethanol und dann in zwei Wäschen von sterilen HBSS.
  4. Entfernen Sie den Kopf vom Körper mit einer Schere. Mit der gleichen Schere, schnitt durch die Haut und den Schädel.
  5. Mit einer feinen Pinzette, schälen den Schädel weg vom Gehirn und Ort des Gehirns in eine kleine Petrischale, dass eine kleine Menge von sterilem HBSS enthält.
  6. Ziehen Sie die Hemisphären. Der Hippocampus ist eine kleine, Seepferdchen-förmige Struktur im medialen Temporallappen.
  7. Entfernen Sie den Hippocampus und in einen 3 ml HBSS in einem 15 ml-Tube. Wiederholen Sie diese Schritte mit jedem Welpen, und legen Sie jeweils isoliert Hippocampus in die 15 ml Tube. Nun ist es Zeit, das Gewebe, einzelne Zellen zu distanzieren.

Hippocampus-Zelle Dissoziation

  1. Nachdem alle hippocampi isoliert wurden, füllen die 15 ml Tube zu 4,5 ml mit HBSS.
  2. Entfernen Sie das Trypsin aus dem Wasserbad, Spray mit Ethanol, und in der Kapuze. 0,5 ml Trypsin auf die Tube, und Inkubation für 15 Minuten bei 37 ° C.
  3. In der Haube, entfernen Sie die HBSS / Trypsin-Lösung aus der Tube mit einer sterilen Pipette, darauf achten, daß der Hippocampus, die den Boden des Röhrchens niedergelassen haben zu stören. 5 ml HBSS auf die Tube und vorsichtig schwenken. Bei 37 ° C für 5 Minuten.
  4. Wiederholen Sie diesen Schritt zweimal, Entfernen der alten HBSS und Ersetzen durch frische Lösung.
  5. Nehmen Sie ein Aliquot von DNAse I aus der Tiefkühltruhe. 0,5 ml DNase zum hippocampi in 4,5 ml HBSS. Die DNAse wird hinzugefügt, um Enzyminaktivierung fördern.
  6. Man reibt die Lösung (oder Pipette nach oben und unten), bis sie homogen ist. Achten Sie darauf, Blasen in das Homogenat einzuführen.
  7. Bestimmen Sie die Lebensfähigkeit der Zellen mit der Trypanblau-Ausschluss-Verfahren.
  8. Nehmen Sie einen 10 cm Petrischale mit 6 bis 8 Deckgläser und 10 ml Plating-Medium. Pipette die gewünschte Anzahl von Zellen, um die Schüssel mit dem Deckgläschen. Vorsichtig zu zerstreuen die Zellen, und stellen Sie sicher das Deckgläschen nicht überschneiden.
  9. Lassen Sie die Zellen für 2-4 Stunden in einer feuchten 37 ° C Inkubator mit 5% CO 2 zu befestigen. Nach der Bestätigung, dass die Zellen lebensfähig sind und angebracht ist, übertragen Sie die Deckgläser auf einzelne Gerichte mit Neurobasalmedium. Legen Sie diese Gerichte wieder in den Inkubator.
  10. Einmal in der Woche, ersetzen ein Drittel des Mediums mit frischem Neurobasalmedium und experimentelle Behandlungen, wie gebraucht. Nun sind die funktionellen Eigenschaften dieser Zellen in Kultur bereit, untersucht werden.

Fura-2 Calcium Imaging von Hippocampus-Neuronen

  1. Nach kultivierten Hippocampus-Neuronen in vitro für 48 Stunden (aber nicht früher) gewesen sein, können Kalzium-Imaging Experimente beginnen. An diesem Punkt sollten diese Neuronen begonnen, Prozesse zu verlängern. Die optimale Altersgrenze für Kalzium-Imaging wird vom ersten Tag an in vitro von 3 bis 7.
  2. Legen Sie Kulturen mit Fura-2 AM für 30 Minuten bei 37 ° C, wonach sie unter einem 20x-Objektiv mit einer Xenon-Bogenlampe, die Kalzium-Indikator-Farbstoff und einem 512 Bit CCD-Kamera, die Proben alle 150 ms erregt abgebildet werden können.

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Discussion

Dieses Protokoll wurde als eine Änderung der grundlegenden Arbeit von Gary Banker und Kim Goslin 1 entwickelt. Dieses Buch ist eine wesentliche Ressource für alle, die Kultivierung von Zellen interessiert - nicht nur das Neuron-angereicherten Kulturen in der aktuellen Protokoll beschrieben.

Die drei wichtigsten Faktoren in der Zellkultur sind: Sterilität, Geschwindigkeit, die Wahl des Mediums verwendet.

Sterilität - eine Laminar-Flow / sterile Haube, aseptischen Bedingungen und einer sterilen Brutkasten benötigt werden. Kontamination bei jedem Schritt schadet nicht nur dem aktuellen Experiment auf dem Sie arbeiten, sondern auch die nachfolgenden Arbeiten, die geplant haben kann. Nach einem Inkubator kontaminiert wird, kann es Wochen dauern (oder ein Monat), um es zu reinigen und steril. Kontamination, die zwar nicht sofort ersichtlich auf eine sichtbare Ebene wird definitiv Auswirkungen auf die Lebensfähigkeit der Zellen und Zellphysiologie. Sterilität ist zwingend erforderlich.

Speed ​​- die Gesundheit der kultivierten Zellen ist stark an die Zeit, die die Zellen nicht in einer Atmosphäre und mittlere kontrollierten Umgebung gebunden. Daher ist es wichtig, dass die benötigte Zeit, um Zellen vom Tier zu entfernen, bis die Zellen in der Beschichtung Medium auf ein Minimum. Da zahlreiche Schritte nicht verändert werden können (time in Inkubation in Trypsin oder gewaschen) werden, was verändert werden kann ist die Menge an Zeit benötigt, um die Zellen aus dem Gehirn zu entfernen. Üben Sie die Dissektion häufig. Die Zeit für diesen Schritt erforderlich muss reproduzierbar sein.

Wahl des Mediums - es gibt eine Vielzahl an geschützten Medien, von denen einer Neurobasal. Vor den Tagen mit Neurobasal, Glia-konditioniertem Medium (die separat gemacht werden musste - dies ist in dem Buch von Banker und Goslin 1 erörtert) erforderlich war. Egal, es ist wichtig, dass Sie wissen, was in dem Medium, das verwendet wird. Zusammen mit Neurobasal, eine Ergänzung, wie B-27 wird oft verwendet. Stellen Sie sicher, dass die Bestandteile dieser mittel-und Nahrungsergänzungsmittel bekannt sind, da sie die Eigenschaften der Zellen beeinflussen können. Es ist gängige Praxis in meinem Labor zu serumfreien / Kohle ausgezogen / Phenolrot-freiem Medium nutzen, um Steroidhormone (vorausgesetzt, dass ein großer Teil der Arbeit in meinem Labor durchgeführt die Aktionen der Steroidhormone untersucht) zu vermeiden. Dieses Konzept - wissen, was in den Lösungen, die Sie verwenden - ist entscheidend für alle Schritte in der Zellkultur.

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Acknowledgments

JLN wurde von MH 68347 unterstützt.

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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