从P0新生大鼠海马神经元的原代培养

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Summary

解剖和个人的大脑区域的细胞的生长,促进细胞和生理参数的调查。我们描述了一个初级细胞培养的方法,在无血清环境产生的神经元丰富的文化。

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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

通常调查海马神经元的生理特性,特别是由于在学习和记忆的海马的参与。海马细胞培养,允许神经学家研究在单个细胞和单突触水平的神经元的活动和属性。在这段视频中,我们将演示如何隔离和成长从新生大鼠海马细胞。海马可能是孤立的,从每个新生动物在短短2至3分钟,和文化可以保持长达两个星期。我们还将简要演示了如何使用这些海马神经元的比例钙成像。虽然这一协议描述为海马的过程中,几乎没有修改,它可以被应用到大脑的其他地区。

Protocol

此前海马隔离

海马隔离开始之前,确保所有的工具是无菌的。喷雾用70%乙醇的文化的油烟机,并置于罩内的工具。您将需要6 - 10厘米的培养皿,无菌聚L镀膜玻璃盖玻片,移液器和技巧,一次性移液器,电动移液器。从这个角度上,记得要使用正确的无菌技术。

  1. 打开水洗澡,并确保它被加热到37℃
  2. 下面的解决方案,保存在4 ° C,将需要:
    • 改良Eagle培养基(MEM)
    • Neurobasal
    • 汉克的缓冲的盐溶液(HBSS)
    • 硼酸盐缓冲液
    • 丙酮酸钠溶液
    • 在蒸馏水中的无菌过滤20%葡萄糖溶液

    • 确保所有的瓶子放置在引擎盖前喷用70%乙醇。

  3. 取出冷冻的这些冻结的解决方案,并放置在一个恒温水浴:
    • 一个马血清5毫升分​​装
    • 一个B - 27的补充1毫升分装
    • 1毫升等分的100X抗生素(青霉素加链霉素)
    • 和L -谷氨酰胺0.5毫升分装
  4. 解冻的解决办法后,取出来的水洗澡,喷用70%乙醇,并将其放置在引擎盖。现在可以准备电镀和Neurobasal媒介。
  5. 的解决方案准备完毕后,盖的容器紧紧地放置在37℃水浴热身,同时执行海马隔离。
  6. 另外,取出的冰柜的蛋白酶胰蛋白酶(2.5%)等分,并放置在水浴。胰蛋白酶消化解剖海马,下一步将在隔离。
  7. 以15毫升锥形管,它填补的HBSS,治疗组和标签。正是在这里隔离海马将在下一步的收集。

海马隔离

  1. 要开始海马隔离,确保新生幼崽是干净的,有他们的牛奶乐队,胎盘,并删除他们的母亲的脐带。
  2. 放置在培养皿中的幼崽,喷用70%乙醇清洗,放置在引擎盖。动物安乐死立即前培养。
  3. 如需进一步消毒,采取一个小狗的菜,沾小狗到70%的乙醇,然后进入无菌的HBSS洗涤。
  4. 删除从身体,用剪刀头。使用相同的剪刀,穿过皮肤和颅骨。
  5. 使用一双细镊子,剥离颅骨远离大脑,把大脑变成一个小型的培养皿中,包含少量无菌的HBSS。
  6. 剥开大脑半球。海马是一个小型的内侧颞叶,海马状结构。
  7. 海马和地点分为3毫升15毫升管的H​​BSS中删除。每个小狗重复这些步骤,每一个孤立的海马放入15毫升管。现在,它是游离于组织单细胞的时间。

海马细胞的解离

  1. 毕竟海马已被隔离,填补了15毫升管4.5毫升的HBSS。
  2. 取出从水浴中,用乙醇喷雾,并在引擎盖的胰蛋白酶。管加入0.5毫升的胰蛋白酶,15分钟在37 ° C。
  3. 在引擎盖上,从管中删除的HBSS /胰蛋白酶液,用无菌吸管,小心不要去打扰的相继落户到试管底部的海马。加入5毫升的HBSS管和涡流轻轻。在37 ° C孵育5分钟。
  4. 重复此步骤两次,删除旧的HBSS,并更换新鲜的溶液。
  5. 以我出的DNA酶等分的冰柜。 4.5毫升的HBSS中加入0.5毫升的DNA酶的海马。添加到促进DNA酶是酶失活。
  6. 磨碎的解决方案(或吸管达上下),直到它被同质。要小心不要引入气泡匀浆。
  7. 与台盼蓝排除法确定细胞活力。
  8. 以10厘米的培养皿中含有6至8盖玻片,并添加10毫升的电镀液。移液器所需的细胞数量的菜含有盖玻片。轻轻地分散细胞的漩涡,并确保盖玻片不重叠。
  9. 允许的细胞附着在饱和湿度37℃培养箱中培养2-4小时,用5%的CO 2 。确认后,这些细胞是可行和有附加,转移盖玻片个别菜肴含有Neurobasal中等。进入孵化器,将这些菜。
  10. 每周一次,更换三分之一的中型新鲜Neurobasal中和试验性治疗,如需要。现在,在培养这些细胞的功能特性加以研究。

FURA - 2的海马神经元钙成像

  1. 后48小时(但不得早) 在体外培养海马神经元钙离子成像实验就可以开始。此时,这些神经元已经开始扩展过程。钙成像的最佳年龄范围是体外3日至7 。
  2. FURA - 2负载的文化AM在37 ° 30分钟,之后,他们可以根据一个20X使用氙弧灯来激发钙指示剂和一个512位的CCD相机,样本每150毫秒的目标成像。

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Discussion

该协议被修改的开创性工作银行家加里和金Goslin 1。这本书是一个重要的资源在培养细胞有兴趣的人 - 不仅在目前的协议描述的神经丰富的文化。

细胞培养的三个最关键的因素是:不育,速度,介质的选择使用。

不育 -层流/无菌罩,无菌条件下,无菌孵化器是必需的。污染不仅不利于目前的实验工作,但也可能已计划的后续工作,在任何步骤。孵化器已被污染后,它可能需要数周(或一个月),清洁和无菌。污染,而不是立即可见的水平明显,肯定会影响细胞活力和细胞生理学。不育是势在必行。

速度 -培养细胞的健康是紧密相连的时间,细胞的氛围和中期控制环境。因此,它是必不可少的,从动物中删除,直到在电镀液中的细胞的细胞所需的时间至少是。因为有许多的步骤不能改变,在孵化中的胰蛋白酶或被冲走的时候,什么可以改变的是删除从大脑细胞所需的时间量。经常练习解剖。这一步所需的时间量需要可重复的。

介质的选择 -有许多专有媒介,其中之一是Neurobasal。天使用Neurobasal,胶质细胞条件培养基(需要单独作出之前-这是讨论的银行家和Goslin 1书)是必要的。无论如何,重要的是你知道什么是在正在使用的介质。随着Neurobasal,如B - 27的一个补充,是经常被使用。确保中期和补充这些成分是已知的,因为它们可能会影响细胞的属性。在我的实验室使用无血清/炭剥离/酚红培养基,以避免类固醇激素(在我的实验室完成的工作,大部份类固醇激素的行为进行调查),它是标准的做法。 - 知道什么是解决方案,您使用的是-这个概念是在细胞培养中的所有步骤的关键。

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Acknowledgments

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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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