P0 Yenidoğan Ratlarda Hipokampal Nöronlar İlköğretim Kültür

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Summary

Diseksiyon ve bireyin beyin bölgesi hücrelerinin büyüme, hücresel ve fizyolojik parametrelere soruşturma kolaylaştırır. Biz nöron zenginleştirilmiş serum serbest bir ortamda kültürler üretir primer hücre kültür için bir metodu tanımlar.

Cite this Article

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Nunez, J. Primary Culture of Hippocampal Neurons from P0 Newborn Rats. J. Vis. Exp. (19), e895, doi:10.3791/895 (2008).

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Abstract

Hipokampal nöronların fizyolojik özellikleri, yaygın olarak, özellikle öğrenme ve hafıza hipokampus katılımı nedeniyle incelenmiştir. İlköğretim hipokampal hücre kültür Nörobilimadamları bireysel hücre ve tek sinaps düzeyinde nöronların aktivitesi ve özelliklerini incelemek için olanak sağlar. Bu video, yeni doğan sıçanlardan alınan primer hipokampal hücreler izole ve büyümek nasıl göstereceğiz. Hipokampus, 2-3 dakika gibi kısa bir her yeni doğan hayvan izole edilmesi ve kültürleri iki hafta kadar devam edebilir. Biz de kısaca bu hipokampal nöronlar oranlı metrik kalsiyum görüntüleme için nasıl kullanılacağını göstermektedir. Bu protokol, hipokampus için süreci tarif ederken, hiçbir değişiklik küçük beynin diğer bölgelerine uygulanabilir.

Protocol

Hipokampal izolasyon önce

Hipokampal izolasyon başlamadan önce tüm araçları steril olduğundan emin olun. % 70 etanol ile kültür başlığı aşağı Sprey, kaputun içindeki araçları. 10 cm Petri kapları, steril poli-L kaplı cam lamelleri, Pipet ve ipuçları, tek kullanımlık pipetler ve elektrikli bir pipet ve - 6 gerekecektir. Bu noktadan itibaren, doğru steril tekniği kullanmayı unutmayın.

  1. Açın ve su banyosunda 37 ° C'ye kadar ısıtılır olduğunu emin olun
  2. 4 saklanır aşağıdaki çözümleri ° C ihtiyaç duyulacaktır:
    • Modifiye Kartal Orta (MEM)
    • Neurobasal
    • Hank tamponlu salin solüsyonu (HBSS)
    • Borat tamponu çözümü
    • Sodyum piruvat çözüm
    • Distile su steril filtrelenmiş% 20 glukoz çözeltisi

    • Kaputun koymadan önce% 70 etanol ile tüm şişe sprey emin olun.

  3. Dondurucu bu donmuş çözümleri alın ve sıcak su banyosu yer onları:
    • 5 ml kısım at serumu
    • B-27 takviyesi 1 ml kısım
    • 1 ml 100X antibiyotik bir kısım (penisilin artı streptomisin)
    • L-glutamin ve 0.5 ml kısım
  4. Çözümleri çözülmüş sonra, su banyosu, onları% 70 etanol ile onları aşağı sprey ve kaput koyun. Şimdi kaplama ve Neurobasal orta hazırlanabilir.
  5. Çözümler hazırlandıktan sonra, hipokampal izolasyon yaparken ısınmak için konteynerler sıkıca kapağı ve 37 ° C banyo koyun.
  6. Ayrıca, dondurucu proteaz tripsin bir kısım (% 2.5) ve su banyosu içine koyun. Tripsin sonraki adımda izole edilecektir disseke hipokampus, sindirir.
  7. Konik tüp 15ml alın HBSS ile doldurun ve tedavi grubu ile etiket. Bu izole hippocampi sonraki adımda toplanan olacağını burada.

Hipokampal İzolasyon

  1. Hipokampal izolasyon başlamak için, yeni doğan yavrular temiz ve süt bantları, plasenta ve göbek kordonu anneleri tarafından kaldırılır vardı emin olun.
  2. % 70 etanol, temiz ve kaput koyun, bir Petri kabındaki sprey yavrular yerleştirin. Hayvanlar kültür hemen önce ötenazi.
  3. Daha fazla sterilizasyon için bir çanak gelen yavru, yavru% 70 etanol içine batırın ve sonra steril HBSS iki yıkar.
  4. Kafası makasla vücuttan çıkarın. Aynı makas kullanarak, cilt ve kafa kesti.
  5. Kafatası beyin kabuğu, bir çift ince cımbız kullanarak ve küçük bir miktar steril HBSS içeren küçük bir Petri kabı içine beyin yerleştirin.
  6. Hemisferlerin geri soyun. Hipokampus, medial temporal lob küçük bir, denizatı şeklinde bir yapıdır.
  7. 3 ml, 15 ml tüp HBSS hipokampus ve yeri içine çıkarın. Her yavru bu adımları tekrarlayın ve her izole hipokampus 15 ml'lik tüp içine yerleştirin. Şimdi, tek hücre doku ayırmak zaman.

Hipokampal hücre disosiasyon

  1. Tüm hippocampi izole edildikten sonra, HBSS ile 4,5 ml, 15 ml tüp doldurun.
  2. Kaputun su banyosu, etanol ile sprey, yer ve tripsin çıkarın. Tüpe 0,5 ml tripsin ekleyin ve 37 az 15 dakika boyunca inkübe ° C
  3. Kaputun, tüpün dibine yerleşmiş hippocampi rahatsız etmemek için dikkatli olmak, steril bir pipet ile tüpten HBSS / tripsin çözüm kaldırmak. Yavaşça tüp ve girdap HBSS 5 ml ekleyin. 37 ° C'de 5 dakika inkübe edin.
  4. Eski HBSS kaldırma ve taze solüsyon ile değiştirerek, bu adımı iki kez tekrarlayın.
  5. Dondurucu dışarı DNAz bir kısım atın. 4.5 ml HBSS hippocampi DNaz 0,5 ml ekleyin. DNAz enzim inaktivasyonu teşvik etmek için eklenir.
  6. Çözüm Karışım (veya yukarı ve aşağı pipetle) homojen kadar. Homojenat kabarcıkları tanıtmak için dikkatli olun.
  7. Trypan mavisiyle boyama yöntemi ile hücre canlılığı belirleyin.
  8. 6 ila 8 lamelleri içeren bir 10 cm Petri kabı alın ve kaplama orta 10 ml ekleyin. Lamelleri içeren çanak için istenilen sayıda hücre Pipet. Swirl hücrelerin nazikçe dağıtmak ve lamelleri örtüşme yoktur emin olun.
  9. Hücrelerin% 5 CO 2 ile nemlendirilmiş bir 37 ° C inkübatör 2-4 saat takmak için izin verin. Hücreler canlı ve ekli olduğunu onayladıktan sonra, Neurobasal orta içeren bireysel yemekleri lamelleri aktarın. Bu yemekleri kuvöz içine yerleştirin.
  10. Haftada bir kez, gerektiği gibi, taze Neurobasal orta ve deneysel tedaviler ile orta üçte birini değiştirin. Şimdi, bu hücrelerin kültür fonksiyonel özellikleri incelenecek hazırız.

Fura-2 Kalsiyum Görüntüleme Hipokampal Nöronlar

  1. Kültürlü hipokampal nöronlar, 48 saat (ama daha önce değil) in vitro sonra, kalsiyum görüntüleme deneyleri başlayabilir . Bu noktada, bu nöronların süreçleri uzatmak için başlamış olmalıdır. Kalsiyum görüntüleme için en uygun yaş aralığı in vitro 3 gün 7 .
  2. Fura-2 ile Yük kültürler, 37 ° 30 dakika AM ° C, 20X hedefi altında örnekleri her 150 ms kalsiyum göstergesi boya ve 512 bit ccd kamera, heyecanlandırmak için bir Xenon ark lambası kullanarak görüntülü sonra.

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Discussion

Bu protokol bir değişiklik Gary Banker ve Kim Goslin 1 yeni ufuklar açan bir çalışma olarak geliştirilmiştir . Bu kitap hücreleri kültür ilgilenen herkes için vazgeçilmez bir kaynak sadece nöron zenginleştirilmiş kültürler geçerli protokol açıklanan.

Hücre kültür en önemli üç faktör vardır: kısırlık, hız, orta seçim.

Laminar akış / Sterilite - steril kaput, aseptik şartlarda, steril bir inkübatör gereklidir. Kirlilik her adımda sadece üzerinde çalışmakta olduğunuz mevcut deney değil, aynı zamanda planlanmış olabilir sonraki çalışma zararlıdır. Bir kuluçka kontamine hale geldikten sonra, temizlenmiş ve steril hafta (veya bir ay) sürebilir. Kirlilik, görünür bir düzeyde hemen belli olmasa da, kesinlikle hücre canlılığı ve hücre fizyolojisi etkileyecektir. Sterilite zorunludur.

Hız - kültür hücreleri sağlık hücreleri bir atmosfer ve orta kontrollü bir ortam değildir süreyi kuvvetle bağlıdır. Bu nedenle, hücreleri kaplama ortamda kadar hayvan hücreleri çıkarmak için gerekli süreyi en azından esastır. Çok adım (tripsin kuluçka veya yıkandıktan zaman) değişmiş olamaz çünkü, ne değişmiş olabilir beyin hücreleri çıkarmak için gereken zaman miktarıdır. Genellikle diseksiyonu Pratik. Bu adım için gerekli süre miktarı tekrarlanabilir olması gerekir.

Orta Seçimi - çok sayıda tescilli ortamlar biri vardır Neurobasal . Neurobasal, glial klimalı ortamda kullanmanın gün önce (ayrı ayrı yapılması gereken bu kitapta Banker ve Goslin 1 tartışılmıştır) gerekli oldu . Ne olursa olsun, kullanılan orta ne olduğunu biliyoruz önemlidir. Neurobasal, B-27 gibi bir ek ile birlikte sıklıkla kullanılmaktadır. Hücrelerin özelliklerini etkileyebilir olarak bu orta ve takviyeleri bileşenleri, bilinen emin olun. Steroid hormonlar (benim laboratuarda yapılan işin büyük bir bölümünü Steroid hormonların eylemleri inceler) önlemek için serum serbest / kömür elimden / fenol kırmızısı ücretsiz orta kullanmak için laboratuvar standart bir uygulamadır. Bu kavram - Kullandığınız çözümler ne olduğunu biliyor hücre kültür tüm adımlar için kritik öneme sahiptir.

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Acknowledgments

JLN MH 68.347 tarafından desteklenmiştir.

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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