小学游离中脑多巴胺鼠类新生儿的细胞培养

Biology

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Summary

小学分离中脑多巴胺的细胞培养允许的多巴胺神经元突触前特性的研究。它们可用于监控实时多巴胺的释放动力学和多巴胺的胞外分​​泌的调节蛋白/ mRNA的水平。在这里,我们将向您展示如何从这些文化灭鼠新生儿。

Cite this Article

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Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates. J. Vis. Exp. (21), e820, doi:10.3791/820 (2008).

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Abstract

允许的孤立的多巴胺神经元突触前特点从大脑中的其它全身投入研究的多巴胺神经元的原代细胞培养创造能力。在我们的实验室,我们使用这些神经元,以评估使用的碳纤维安培,以及使用定量PCR和免疫细胞化学的多巴胺相关基因和蛋白质表达水平的多巴胺的释放动力学。在这个视频中,我们将向您展示我们如何产生这些文化灭鼠新生儿。

这一过程涉及几个步骤,包括电镀皮层胶质星形胶质细胞,神经细胞的培养基由胶质基质空调,中脑新生儿的解剖,消化,提取和多巴胺神经元的电镀和神经营养因子除了,确保细胞的存活。

应用适合这样的准备,包括电生理学,免疫细胞化学,定量PCR,视频显微镜(即实时与质膜融合水泡),细胞活力检测和其他毒理学屏幕。

Protocol

制备前文化

:**神经胶质细胞,必须提前做好,让他们有时间,增殖和覆盖的菜肴底部镀。对于非典型的或实验的遗传背景的小鼠,确保胶质细胞和神经元的文化相匹配的基因型。

:** 1-7天前清扫,准备好新鲜的神经介质,并取代在用2ml的菜的胶质介质。

:**在清扫前的一天,以下项目需要紫外光下过夜左:

  • 4夹层板
  • 2解离样品瓶与一个微型磁力搅拌棒
  • 2小瓶的瓶盖(2小孔,顶部探出)
  • 摊开幻灯片环(和70%乙醇外套) - 离开盒也开放
  • 至少2黄(10 -200μL)枪头盒
  • 1盒蓝色(100 -1000μl)移液器

文化日

:*千万不要碰任何东西离开紫外线在外过夜!

引擎盖下保持无菌的环境是非常重要的的。

1)设置:

:**抛开所有冻结的成分,使木瓜蛋白酶溶液。

  1. 用70%乙醇的清洁钳。盖,用无菌的黄色吸管尖每个提示。使用放置在无菌幻灯片戒指的盒子。
  2. 热点板块:
    1. 将下一个千毫升烧杯装500 -600毫升DH 2 O和一个大的磁力搅拌棒的引擎盖mini-magnetic/hot板。
    2. 水位以上的烧杯放置在一个泡沫塑料盘。
    3. 将在泡沫塑料盘的小孔中的温度计。热拨号需求将略高于2(温度为34℃)和搅拌转盘应〜5-6。
  3. PBS:
    1. 准备无菌PBS,填补4-6毫升15无菌15毫升管(一定要保持无菌
      技术)。
    2. 管和股票瓶放在旁边的引擎盖上冰。
  4. Carbogen:
    1. 打破了一半5毫升stripette,推动通过一个橡胶塞,所以stripette的一角伸出广角端的塞子。
    2. 400 - 500ML的DH 2 O(stripette断头应在DH 2 O),放置在一个500ml烧瓶的瓶塞。
    3. 将烧瓶的侧开管。
    4. 切过一个黄色的枪头的提示和连接管端(切端朝外)。

      连接carbogen坦克5毫升stripette的一角。

  5. 准备木瓜sol'n:
    1. *使用注意无菌技术。
    2. 混合在无菌的50毫升管。
  6. 过滤木瓜sol'n到解离小瓶:
    1. 25毫升stripette(或地方新上一个20毫升柱塞注射器的尖端0.2微米的注射器过滤器通过使用一个Steriflip过滤单元和转移解离小瓶,
      取出柱塞和使用stripette(25毫升)的注射器转移到所有的木瓜蛋白酶sol'n。
    2. 解离瓶过滤器)。
    3. 第小瓶,在第一个通过孔插入无菌注射器和附加0.2μm的无菌注射器过滤器,注射器的基础。
    4. 连接的carbogen过滤器和封口膜这个地方。放置在发泡胶盘/持有人的小瓶。

      *必须移动的微型磁力搅拌棒。
      *气体必须到小瓶。
      *温度必须稳定在34 ° C。

  7. 夹层的准备:
    1. 将发动机罩下的解剖镜下。
    2. 莱铝箔所有清扫工具,喷以70%乙醇,倒出多余的和干引擎盖下离开。
  8. 准备神经介质:
    1. 将新鲜的神经介质的孵化器30毫升。
  9. 莱1铝箔的大型广场和12个小方格。保留铝箔斩首剪刀。用冰水填写一个小的发泡胶盒,并保留所有罩外。
  10. 准备麻醉(氯胺酮和0.075毫升0.075毫升xzylazine)。
  11. 100板,至少有12个幼崽(P0 - P2的)。对小鼠,确保整个垃圾的基因型有一个匹配。如果基因型是未知的,从每一个单独的盘上的小狗的板块细胞,保持准确的记录鼠标号码和与之相配的细胞培养皿,确保胶质基质的遗传背景是在细胞培养的统一。

2)解剖

  1. 腹腔注射麻醉的第一个动物。动物表演时的镇静和不响应甩尾测试;在冰为30秒(直到低温)。冲洗一个小铝箔平方米,开盖itation剪刀,并用70%乙醇的负责人。
  2. 杀头,让头落在铝箔广场和引擎盖下移动。轻轻地取出成一个冰冷的PBS15毫升管的大脑。广场上冰管,同时消除未来的大脑。
  3. 重复这些步骤,直到有3个冰大脑。在显微镜下的第一个夹层菜倒到所有3个大脑和PBS。删除大脑中的相应部分(VTA)和使用转移吸管放入木瓜sol'n段。第一分部进去时,启动一个计时器
  4. 重复以前的3个步骤,直到所有的大脑正在木瓜蛋白酶消化。平均消化时间应在2小时。第一部分已在最后进去的时间约1小时

    尝试分裂的区别。

  5. *在消化过程中:
    1. 确保在洗澡的温度是34 ° C。
    2. 段可能会粘在第一个搅拌棒,但随着时间的推移应该传播出去。如果他们不这样做,挖掘瓶底部。

3)Trituration:

  1. 使用移液管,转移大脑段无菌15毫升管(尽量避免多余的木瓜蛋白酶sol'n),并把它们洗干净回暖胶质介质从孵化器2毫升3倍。将在管内的胶质介质后,允许部分解决和不扰民的细分,然后小心地取下尽可能sol'n。更改移液器,以避免污染!

    **不要让被删除sol'n。

  2. 开始使用一个新的移液管中的胶质媒体2毫升triturations。磨碎的25倍(避免气泡出租),并让管坐3分钟,直到未解离段解决。使用移液管,以消除和尽可能保持尽可能多的sol'n互不干扰段底部。保存在一个新的(无菌)15米管上清液。
  3. 重复上一步一个1000μl移液器和200μL的移液器使用,直到完全分离。
  4. 磨碎用移液管的10倍,或待细胞完全悬浮在sol'n。

4)电镀细胞

  1. 使用无菌黄色枪头,覆盖每个镊子尖,仔细下降为中心的玻璃内的每一道菜,以及可能的幻灯片环。
  2. 放在hemacytometer和计数的细胞液中加入10μl。 (不包括垃圾群众)。
  3. 乘以10的细胞数量。这是细胞数/μL。这个数字除以1,000,000-1,500,000(或想要的密度)。这是μL每道菜添加的数量。
  4. 使用枪头滑过每道菜的中心,以及戒指,当你添加适当μL/碟。钢板他们轻轻地切换为每道菜的提示。
  5. 加入100μL(稀释)GDNF的每道菜的幻灯片环内sol'n。
  6. 此时,放置在孵化器的托盘,并带来了在寒冷的房间里的神经介质。
  7. 使细胞一夜之间解决。
  8. 第二天:删除环(使用新的无菌枪头,覆盖每道菜的镊子提示)

5)有丝分裂的抑制作用:第二天

  1. 加入200μLFDU股票1.8毫升无菌的MEM稀释1000X股票1:10 FDU等分。
  2. 加入20μL稀释FDU sol'n每道菜的外部环。经常更改枪头。封面和返回的孵化器。

    *打扰尽可能少地为未来7-10天的菜肴。检查感染的菜肴和删除任何受感染的菜肴,在感染的最初迹象。文化3个星期内测试的准备。

媒体/解决方案

神经元的中等

(200ML)

对神经胶质细胞**最佳,如果空调使用前洗/ trituration一夜之间从烧瓶

成分 金额 注释
BSA的5% 0.5摹分数V
纪念液体 94.0毫升西格玛
DMEM培养液 80.0毫升西格玛
F - 12液体 20.0毫升西格玛
葡萄糖45%液体 1.50毫升六西格玛解决方案
谷氨酰胺200MM 0.5毫升 Aliquotted六西格玛解决方案
Diporzio浓。 2.0毫升六西格玛解决方案
液体过氧化氢酶 0.1毫升
Kynurenic酸0.5M 200μL 在1N的NaOH
盐酸5N 50μL

  1. 结合在250米成分(牛血清白蛋白进入最后)升塑料瓶。
  2. 过滤器,标签和冷藏。

Kynurenic酸
(FW = 189.2)
0.5M = 94.6mg/ml
设为8毫升股票:无菌分装到200μL756.8mgKA/8ml 1N氢氧化钠和吸管



DiPorzio媒体

一)DiPorzio浓。股票:

必要性 结合 Alliquot
添加剂 溶剂 金额 毫升 毫升/管 浓。 金额 #Aliq。
胰岛素 20mm的盐酸(1) 塑料 250毫克瓶 10 1 25mg/ml 25毫克 10
汉克的 500毫克瓶 5 1 100mg/ml 100毫克 5
超氧化物歧化酶汉克的 70mg瓶 14 1 5mg/ml 5毫克 14
腐胺汉克的 50毫克 3 0.12 20mg/ml 2.4mg 21
NA 2 SEO 3 汉克的 0.104mg 10 0.5 10μg/ml 5.2μg 20
T3 10MM的NaOH 2毫克 10 0.1 0.20mg/ml 0mg 100
孕酮 100%乙醇玻璃 12.5mg 10 0.05 1.25mg/ml 使用吸管
皮质醇 100%乙醇玻璃 20毫克 10 0.02 2.00mg /毫升使用吸管

  1. 20MM盐酸=41.5μl浓度。 HCl/25ml。
  2. 1mg/ml的股票,并添加104μl至10ml。



二)DiPorzio媒体股票:

添加剂 数量(毫升) 金额(毫升)X2 终浓度。微克/毫升 终浓度。克分子浓度
孕酮 0.05 0.1 0.06 200nm的
皮质醇 0.02 0.04 0.04 125nM
汉克的的BSS 6.21 12.42
胰岛素 1 2 25
NA 2 SEO 3 0.5 1 0.01 30NM
T3 0.1 0.2 0.02 30NM
超氧化物歧化酶 1 2 5
腐胺 0.12 0.24 2.4 15NM
1 2 100

  1. 使用无菌15毫升聚丙烯管添加孕激素和皮质醇。使用吸管抽吸和真空中速度的乙醇蒸发:(使用5毫升stripette一半,中途破入管非常小心,不要吸乙醇液体安全保持Kimwipes地方吸管,然后将小费。 500μL商标管一侧。
  2. 他们在上面的图表中出现的顺序添加其后等分。
  3. 除了胰岛素,这使得sol'n阴天后,加1N氢氧化钠20μL中的pH值。 Sol'n应该从黄色变为粉红色,立即清除。此外,除了后转,立即加入20μL1N氢氧化钠中的pH值和防止沉淀物的形成。
  4. 绘制成血清吸管和划分成5等份(一批)的毫升。
  5. 商店@ -20 ° C。
  6. 五月2批一次。

解离媒体

A.木瓜蛋白酶(1瓶):

**电镀准备清扫前一天。

成分 金额</ STRONG> (注)终浓度。
半胱氨酸水 7.8mL 1MM半胱氨酸
木瓜蛋白酶变化(瓶*190μl
44.0mg/ml)
20单位/毫升(共400个单位
20毫升值得sol'n)
H&B浓度。 2ML
Carbogen 95%O2,5%CO2
盐酸5N 加入10μl
0.5%酚红 20μL 0.001%
Kynurenate 0.5M 加入10μl (1N氢氧化钠)0.5MM

  1. 添加木瓜蛋白酶半胱氨酸水。
  2. H&B浓度,kynurenate,盐酸,酚红。
  3. 过滤器分解小瓶(设置方向),并连接到carbogen。


B. H&B浓缩液(100毫升的5倍)

配料 兆瓦 Powder/50ml H 2 O 浓。 (M) 联合收割机(毫升) 终浓度。 (MM)
氯化钠 58.44 11.699克 4 14.5 116
氯化钾 74.56 3.728克 1 2.7 5.4
碳酸氢钠3 84.01 4.2摹 1 13 26
的NaH 2 PO 4 · H 2 O 137.99 6.90摹 1 1 2
硫酸镁 120.38 6.019克 1 0.5 1
EDTA(ED2 - SS)
292 Sigma公司5%(请
5g/100ml股票)
0.134 0.3722 0.5
血糖 180 适马45% 2.5 5 25
TC H 2 O 62.93

  1. 结合股票的解决方案的金额。
  2. 分成4毫升等分。
  3. 新增分装后,加入木瓜蛋白酶半胱氨酸水。

C.半胱氨酸水(1X157.5毫升):

配料 兆瓦 组件
粉(毫克)
H 2 O(ML) 股票浓度(MM) 联合收割机(毫升) 终浓度。 (MM)
氯化钙 147.2 736 10 500 0.6 为1.9mm
半胱氨酸 121.7 (1.5) 24 20MM(0.02M) 10 1.27MM
(0.88)
训练班水 146.9

  1. 请和的0.5M氯化钙股票sol'n保持在4 ° C
  2. 与其他成分的半胱氨酸使用sol'n并结合
  3. 15.75毫升和存储分为10等分,分频在4 ° C


GDNF的制备

  1. 分装准备:
    1. 灭菌去离子水溶解2.4mL无菌,冻干颗粒5μgGDNF的。本GDNF的sol'n =2.08μg/mL浓度。
    2. 分配到76.9μl在无菌离心管分装2.08μg/mLGDNF的sol'n。这每小瓶= 160ng。
    3. 储存在-20 ° C。
  2. 除了文化:
    1. 解冻为每8菜1整除。
    2. 加入723.1μl神经介质/等分稀释分装。这将使160ng/800μlGDNF的sol'n。
    3. 这sol'n100μL加入终浓度为10ng GDNF的每毫升的媒介,在每道菜2ML。


复旦的制备

FDU - sol'n 1000X股票:

成分 金额 (注)终浓度。
尿苷 247毫克 16.5毫克/毫升
5 FDU(5 fluorodeoxyuridine) 100毫克(瓶) 6.7毫克/毫升
训练班水 15毫升

  1. 超过15毫升16.5毫克/毫升尿苷一点点。
  2. 添加15毫升尿苷sol'n每瓶100毫克的FDU 6.7毫克/毫升sol'n FDU。
  3. 在-20 ° C到200μL分装和冻结的鸿沟

稀释使用:

  1. 抑制非神经元细胞的生长。加入200μL的股票,以1.8毫升的MEM稀释1000倍的股票1:10。
  2. 加入20μL稀释FDU每道菜的外部环。经常更改吸管提示。封面和返回的孵化器。

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Discussion

这里所描述的方法,让精细的分辨率在全身或可在体内的方法,否则不中枢多巴胺神经元的形态和神经化学物质的功能。

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Acknowledgements

这项工作是支持DK065872(ENP),一个卓越的史密斯家庭基金会在生物医学研究奖(ENP),F31 DA023760(BMG,ENP)和P30 NS047243(塔夫茨大学神经科学研究中心)。

Materials

Materials are included in the protocol text.

DOWNLOAD MATERIALS LIST

References

  1. Geiger, B. M., Behr, G. G., Frank, L., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., Pothos, E. N. Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats. FASEB Journal. 8, 2740-2746 (2008).
  2. Pothos, E. N. Regulation of dopamine quantal size in midbrain and hippocampal neurons. Behavioural Brain Research. 130, 203-207 (2002).
  3. Pothos, E. N., Larsen, K. E., Setlik, W., Gershon, M. D., Krantz, D., Liu, Y. -J., Edwards, R. H., Sulzer, D. Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size. The Journal of Neuroscience. 20, 7297-7306 (2000).
  4. Pothos, E. N., Davila, V., Sulzer, D. Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size. The Journal of Neuroscience. 18, 4106-4118 (1998).
  5. Sulzer, D., Pothos, E. N. Presynaptic mechanisms that regulate quantal size. Reviews in the Neurosciences. 11, 159-212 (2000).

Comments

29 Comments

  1. Excellent!

    Reply
    Posted by: Anonymous
    November 14, 2008 - 11:03 AM
  2. I would like to thank Lauren and Emmanuel for such an excellent piece of science. I was thinking of setting up the method in my lab and the video encouraged me to do so. Sure it helps a lot!!! Hope to see more like this video in the future.

    Reply
    Posted by: Anonymous
    January 13, 2009 - 11:59 AM
  3. Thank you very much for this wonderful protocol. I am very excited to try this out. Could you please let me know what kind of plates you used for plating the glia and where did you buy the slide rings from (catalog number perhaps). Thanks a bunch!

    Reply
    Posted by: Anonymous
    February 23, 2009 - 4:38 PM
  4. Thank you for the comment and we are happy to help. The dishes were ordered from World Precision Instruments: FlouroDish FD35PDL-100, tissue culture dish with cover glass bottom, dish:35mm glass:²3mm. Thanks, Lauren

    Reply
    Posted by: Anonymous
    April 23, 2009 - 11:20 AM
  5. And slide rings: Thomas 6705-R1² Thanks.

    Reply
    Posted by: Anonymous
    April 23, 2009 - 1:57 PM
  6. can you please tell me how to separate astrocyte from neurons using serum coating. please send a protocol if possible. thanks Gaurav jain gauravjain_niper@yahoo.co.in

    Reply
    Posted by: Anonymous
    April 17, 2009 - 10:27 PM
  7. We do not have a protocol to separate astrocytes from neurons, all our cell sorting is done through immunocytochemistry on fixed tissue. Best, Emmanuel

    Reply
    Posted by: Anonymous
    April 24, 2009 - 3:21 PM
  8. I have never worked with midbrain coltures and I really appreciate your video that will help me for sure.
    I have a question, when you first plate astrocytes ² weeks before, you mean astrocytes from midbrain tissue? Or maybe are they from cortex or hyppocampus? Because I know how to prepare cortical astrocytes but I would like to know if it is possible to prepare them also from midbrain.
    Thanks in advance.

    Ilaria

    Reply
    Posted by: Ilaria V.
    November 15, 2009 - 4:18 AM
  9. Cortical astrocytes are the best option as the midbrain dŒs not provide as high a yield. Best, Emmanuel

    Reply
    Posted by: Anonymous
    November 15, 2009 - 7:44 AM
  10. I have never worked with midbrain coltures and I really appreciate your video that will help me for sure.
    I have a question, when you first plate astrocytes ² weeks before, you mean astrocytes from midbrain tissue? Or maybe are they from cortex or hyppocampus? Because I know how to prepare cortical astrocytes but I would like to know if it is possible to prepare them also from midbrain.
    Thanks in advance.

    Ilaria

    Reply
    Posted by: Ilaria V.
    November 15, 2009 - 4:28 AM
  11. Hi, the astrocytes we derive from cortical tissue (higher yield than in the midbrain).

    Reply
    Posted by: Anonymous
    February 18, 2010 - 1:14 PM
  12. Hi there,

    It's great to find this info online, many thanks for submitting it. Can I ask what kind of % DA neuron yields you're getting this way?

    Cheers,

    Alex

    Reply
    Posted by: Alexander J.
    February 18, 2010 - 9:39 AM
  13. The yield is in the range of 40-60% if cells are plated in the presence of GDNF. Otherwise, the yield is in the range of 10-²0%.

    Reply
    Posted by: Anonymous
    February 18, 2010 - 1:17 PM
  14. Hi again,

    Sorry, i had one other thing to ask if I may. We have a lot of experience with hippocampal dissociated cultures and would be interested in seeing whether the midbrain cultures can work with the media we use for these (Neurobasal and variants); in trying this, is there any specific nutritional or medium requirement of DA neurons you've identified that we should be aware of, which isn't likely to be something we are already giving the hippocampal neurons? I see you include things like catalase and kynurenic acid that aren't in our medium......I presume these are to minimise oxidative and excitotoxic stress? Can i ask, do you have an idea of how much these improve the yield by? Many thanks again, Alex

    Reply
    Posted by: Alexander J.
    February 18, 2010 - 10:36 AM
  15. As mentioned above, the single most critical factor in improving dopamine neuron yield is GDNF.

    Reply
    Posted by: Anonymous
    February 18, 2010 - 1:19 PM
  16. Many thanks indeed for this information, that's really helpful. cheers,
    Alex

    Reply
    Posted by: Anonymous
    February 18, 2010 - 4:46 PM
  17. Sorry, one final one from me - have you ever tried the Banker method with these? I have heard this dŒsn't work as well as having the cell layers in full contact, is this your experience? Is it very much worse? I am very interested in trying Banker because a neuronal monolayer would work much better with the microscopy set up I want to use! Many thanks, Alex

    Reply
    Posted by: Alexander J.
    February 19, 2010 - 4:59 AM
  18. No, we have not tried the Banker protocol on this.

    Reply
    Posted by: Anonymous
    February 19, 2010 - 12:25 PM
  19. Hello, I wonder if I can ask what you coat your culture dishes with before plating astrocytes? I am finding that mouse glia may be quite sensitive to the usual things like poly-D-lysine etc. Many thanks, Alex

    Reply
    Posted by: Anonymous
    April 8, 2010 - 9:01 AM
  20. Poly-D-lysine is fine for rat astrocytes, laminin is better for mouse astrocytes (although poly-D-lysine has also worked for us in mouse cultures).

    Reply
    Posted by: Anonymous
    April 28, 2010 - 2:56 PM
  21. Hi, thanks for the protocol. Can you explain why you use P0-P² mice? I am looking to study early DA neurons from what would become the VTA and was planning on using embryonic brains, so wondered what the reason was for using neonatal brains here.

    Many thanks,
    Naomi

    Reply
    Posted by: Naomi P.
    October 4, 2012 - 10:29 AM
  22. Using embryonic instead of neonatal brains with this procedure would be fine provided that you account for adequate brain development in utero. To be safe and ensure health of the dopamine cell bodies in culture, I would avoid using embryos prior to the third trimester of gestation. We have not used dissociated cultures for precursors to dopamine neurons so I am not really qualified to vouch for the viability of such preparations. Emmanuel

    Reply
    Posted by: Anonymous
    October 18, 2012 - 6:55 PM
  23. Ok, thank you!

    Reply
    Posted by: Naomi P.
    October 19, 2012 - 4:20 AM
  24. Hi Naomi, the protocol should work with embryonic brains as well (provided you allow for development of DA neurons) but the dissection is going to be much more challenging. I would suggest you culture neurons from the midbrain (both VTA and nigra) in that case to allow for the difference in size of the brain sites you are targeting. MIcrodissection techniques may also help, but you have to find the way to get your tissue in carbogenated media ASAP.

    Reply
    Posted by: Anonymous
    November 20, 2013 - 12:44 PM
  25. Thank you for posting such a great informational video. I have a follow-up question to the previous question on this page: would it also be appropriate to use postnatal day 2-5 mice instead of PN 0-2? I ask because I have seen other protocols to culture primary dopamine neurons that use older mice (for example, Smeyne and Smeyne, 2002).

    Thanks,
    Brittany W.

    Reply
    Posted by: Brittany W.
    July 5, 2013 - 2:18 PM
  26. Hi Brittany, you can try with P2-P5 but it is not optimal. P0-P2 would typically give you the highest yield in live DA cells in culture, Emmanuel

    Reply
    Posted by: Anonymous
    November 20, 2013 - 12:47 PM
  27. Thanks!

    Reply
    Posted by: Brittany W.
    November 21, 2013 - 2:39 PM
  28. Hi, The protocol is explained and shown very nicely. I want to do only dopaminergic neuron culture. Is it possible to do the culture without glia or seeding it directly on poly d lysine coated plates. In your protocol you have added FDU to kill dividing cells (glia), does it kill all within and outside the ring in the plate, and you get only dopminergic cells at the end. Would you please help and suggest a bit. Thanks. Farah

    Reply
    Posted by: Farah S.
    November 20, 2013 - 10:27 AM
  29. Hi Farah, we have not been able to produce DA cell cultures without plating glia, the latter are needed to condition the media and help sustain the culture for several weeks. If you only think of acute experiments, it might be possible to isolate DA cells for a few hours, but they would have no neurites, they would be difficult to identify as DA neurons and they would die very fast. Best, Emmanuel

    Reply
    Posted by: Anonymous
    November 20, 2013 - 12:51 PM

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