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Hippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers
海马体神经的分离转化以及在微腔中的培养   

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Abstract

Microfluidics chamber is an ideal tool to study local events that occurring in neuronal projections by perfectly compartmentalizing the cell soma from certain branches. It is very well suited for live cell imaging or immunohistochemistry staining. This protocol has been carefully modified in detail to fit the requirement of primary rat hippocampal neuronal cultures. It can also be applied to a more general neuronal culture purpose in microfluidics.

Materials and Reagents

  1. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  2. HBSS (Hyclone, catalog number: SH30268 )
  3. Boric acid (Sigma-Aldrich, catalog number: B0252 )
  4. Borax (Sigma-Aldrich, catalog number: B9876 )
  5. Cysteine (Sigma-Aldrich, catalog number: C7352 )
  6. EtOH
  7. Neural basal media (NBM)
  8. Glutamax
  9. B27
  10. PenStrep
  11. Gentamicin
  12. Glutamate
  13. FBS
  14. Laminin
  15. Trypan blue
  16. Na2SO4
  17. K2SO4
  18. MgCl2
  19. CaCl2
  20. Glucose
  21. Phenol Red
  22. EDTA
  23. 0.1 M borate buffer (pH 8.5) (see Recipes)
  24. Dissection media (DM) (see Recipes)
  25. Papain activation buffer (see Recipes)
  26. Papain (100 mg) (Worthington Biochemical Corp, catalog number: LS003119 ) (see Recipes)
  27. DNase (F. Hoffmann-La Roche, catalog number: 10104159001 ) (see Recipes)
  28. Papain digestion media (see Recipes)
  29. Dissociation media (see Recipes)
  30. Plating media with phenol red (see Recipes)
  31. Growth media without phenol red (see Recipes)

Equipment

  1. PDMS devices
  2. Microscope
  3. Hemacytometer
  4. TC hood
  5. Incubator
  6. -80 °C freezer
  7. 37 °C water bath
  8. 6 cm cell culture dishs
  9. Plasma-bonding machine (model PDC-32G , http://www.harrickplasma.com/)

Procedure

  1. Coverslip and Chamber Preparation
    Day 1:
    1. PDMS devices are fabricated as described in Park et al. (2006).
    Day 2:
    1. Corning No.1, or No.1.5 coverglasses are both suitable for our microscope (24 mm x 40 mm). Or, Carolina coverglasses 24 mm x 50 mm.
      1. Sonicate the cover glasses in autoclaved ddH2O for 60 min.
      2. Sonicate again in 70% EtOH for 30 min.
      3. Sonicate in 100% EtOH for 30 min.
      4. Wash with autoclaved ddH2O in TC hood, air dry the coverglasses overnight in the hood.
    Day 3:
    1. Assemble the coverglass and PDMS device by using the plasma-bonding machine.
    2. Press firmly on the corners but only lightly touch the middle channel. Coat the chamber with PDL (>150K mol wt, prepared in borax boric acid buffer):
      1. 0.5 mg/ml PDL is added to a well on one side of the device and allowed to flow through into the connecting well (leave the other side unfilled).
      2. Allow the PDL to flow through the device for about 10 min and then add more PDL to the wells to fill them up.
      3. Place the devices containing PDL in an incubator at 37 °C for a minimum of 30 min to let the PDL fill the micro-groves.
      4. Fill the wells and major channel on the other side of the chamber with PDL and return the chamber to incubator at 37 °C for a minimum of 4 h, overnight is preferable.
    Day 4:
    1. Pipette out the excess PDL after treatment, but be careful not to suck all the liquid out of the device.
      1. Add autoclaved ddH2O to each well on either side of the device, and allow to flow to the other well by capillary action.
      2. Aspirate off the water (again being careful not to fully remove all the liquid from the device), then add another 150 μl of autoclaved ddH2O to one of each connected well and allow it to flow through to the corresponding well.
      3. Repeat this quick wash twice, then place the device containing ddH2O in an incubator at 37 °C for 1 h.
      4. Then, repeat the wash step again. Wash the devices at least three times with ddH2O for one hour each, which is the total.
      5. Add NBM with the necessary factors (glutamax, B27, PenStrep) to the top wells. Let it flow through, incubate at 37 °C overnight.
      6. Prepare 40 ml plating media:
        800 μl B27 + 30 μg/ml gentamicin + 400 μl glutamax + 25 μM glutamate
      7. Prewarm 10 ml dissociation media in 15ml tube: Neurobasal + 5% FBS + gentamicin.
      8. Prewarm 10 ml plain neurobasal media for papain.
    Day 5:
    1. Add laminin (20 μg/ml) to each well and incubate at 37 °C for at least 30 min: Coat the chamber with laminin: take a 20 μl aliquot out from -80 °C freezer and put it on ice immediately, wait until it dissolves completely on ice then add cold neurobasal media to 1.8 ml.
      1. Turn the UV light on and UV sterilize one 6 cm cell culture dishs for 15 min.
      2. Pre-warm 14 ml plating media in this 6 cm cell culture dish, 37 °C incubator.
      3. Replace the laminin solution with plating media 1 h before plating the cells.

  2. Dissection of hippocampal neurons
    1. Activate papain 10 mg aliquot with 750 μl papain buffer at 37 °C for 30 min.
    2. Add to prewarmed neurobasal media, filter sterialize, then add one DNase 20 μl aliquot.
    3. Wash the tissue with 10 ml cold dissection media in TC hood (wash twice).
    4. After the tissue settled by gravity, remove HBSS completely.
    5. Add one aliquot of DNase to 10 ml digestion media (prewarmed yesterday).
    6. Place half of digestion media (5 ml) onto the tissue for 15 min at 37 °C water bath.
    7. Add the remaining half of digestion media into the tube and incubate for another 15 min at 37 °C water bath.
    8. Let the tissue settle to bottom by gravity or spin shortly.
    9. Re-suspend the pellet with 5 ml papain dissociation media and triturate up and down with 5 ml pipette for 5 times, avoid bubbling the media.
    10. Let debris settle slowly, or short spin to 100 g up and down (600 rpm), transfer the supernatant to a new 15 ml tube.
    11. Continue triturate the undissociated tissues for 5-10 times, take the supernatant and combine with previous 5 ml.
    12. Take 10 μl out and mix with 10 μl trypan blue.
    13. Load 10 μl mixture on hemacytometer and count:
      All cells in 16 small squares x 104 = cells/ml (A typical yield from 12 E18 pups is ~50 M in total).

  3. Amaxa transfection
    1. Mix 82 μl nucleofector solution with 18 μl supplement before use.
    2. Prepare 50 μl nucleofector with 5 μg total DNA (if using more than 1 plasmid, mix in equimolar ratios).
    3. Spin down 5 million (1-6 million for each transfection) neurons at 150 x g 5 min. During spinning, transfer 7 ml pre-warmed plating media to a 15 ml tube, add 350 μl FBS, keep at 37 °C. Prepare the 1 ml pipette with a tip.
    4. After spinning, remove all media using aspiration pipette with a 200 μl pipette tip.
    5. Resuspend the cells in a 50 μl nucleofector solution.
    6. Mix 50 μl of the cells with the premix of DNA + neucleofector (a total of 100 μl).
    7. Zap with program G-13 (better for Hc) or O-03 (better for Cx). A foam of dead cells will form.
    8. Immediately pipette in 1 ml warmed plating medium with FBS and pipette out to a 1.7 ml eppendorf tube. Do not pick up dead foam. Be gentle.
    9. Incubate at 37 °C 10 min after rescue.
    10. Spin down the cells at 150 x g, 5 min.
    11. Resuspend in 300 μl plating media with FBS.
    12. Load 5 μl/well, filling the two upper wells of each microfluidic.
    13. Let cell adhere at 37 °C for 10 min.
    14. Fill the wells from both ends simultaneously with two pipettes with plating media with FBS.
    15. Completely change media to fresh plating media W/O FBS 2 h later.
    Day 6:
    1. Change media once again the next morning with plating media.
    2. Change half of the media every 2 days.

Recipes

  1. 0.1 M borate buffer (pH 8.5) (borax boric acid buffer)
    1.24 g Boric acid
    1.9 g Borax
    Bring to 500 ml with ddH2O
    PH should be 8.5 without adjustment
    Filter sterilize
    Store at 4 °C.
  2. Dissection media (DM) (for 1L)
    82 ml 1 M Na2SO4
    60 ml 0.5 M K2SO4
    5.8 ml 1 M MgCl2
    252 μl 1M CaCl2
    8 ml 2.5 M glucose (20 mM)
    1 ml 1 M Hepes (pH 7.4)
    2 ml 0.5% Phenol Red pH with 1 M NaOH to 7.4 filter sterilize and keep at 4 °C.
    OR just use HBSS and add glucose to 20 mM, HEPES:
    500 ml HBSS with Ca2+ and Mg2+
    5 ml 1.5 M D-glucose (filter sterilized)
    5 ml 1.0 M HEPES pH 7.2
  3. Papain activation buffer (50 ml)
    1.1 mM EDTA 110 μl (0.5 M EDTA, pH 8.0)
    0.067 mM β-mercaptoethanol 0.25 μl (0.0047 μl BME/ml buffer)
    5.5 mM cysteine 33.25 mg
    Store at 4 °C.
  4. Papain
    Aliquot into 10 mg/1.7 ml eppendorf tube, Store lyophilized powder at 4 °C
    Add 750 μl papain buffer before use
    Activate to 200 U/ml (10 x) - 30 min at 37 °C (shake the tube once in a while)
  5. DNase
    1,000 U/μl stock, 20 μl aliquot (20,000 U/tube, used in 10 ml)
  6. Papain digestion media (for every ten animals)
    Add the 750 μl activated papain to plain neurobasal media
    Filter sterilize
    Add DNase: 2 U/μl DNase
  7. Dissociation media (in 10 ml neurobasal)
    Gentamicin 40 μg/ml
    FBS 500 μl
  8. Plating media with phenol red (in 20 ml neurobasal) with serum
    B27 400 μl (Gem21, NS21 all the same)
    Gentamicin 40 μg/ml (10 mg/ml stock, take 80 μl/20 ml media)
    Glutamax 200 μl
    Glutamate 25 μM (100 mM stock, 4,000x, 5 μl/20 ml) (use at 12.5 μM final concentration may be better: 2.5~3 μl/20 ml)
    FBS 200 μl
  9. Growth media without phenol red (in 20 ml neurobasal)
    B27 400 μl (Gem21, NS21 all the same)
    Glutamax 200 μl

Acknowledgments

Alternative reference protocol can be found on the Millipore website related to AXISTM Axon Isolation Devices. The dissection protocol is modified from a protocol developed in Dr. Michael Lin’s lab, Department of Pediatrics and Bioengineering, Stanford University, USA.

References

  1. Detailed reference protocols can be requested from http://www.xonamicrofluidics.com/about.html.
  2. Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W. and Jeon, N. L. (2006). Microfluidic culture platform for neuroscience research. Nat Protoc 1(4): 2128-2136.

简介

微流体室是一个理想的工具,研究发生在神经元投射的本地事件,通过完美地划分细胞体细胞从某些分支。 它非常适合活细胞成像或免疫组织化学染色。 这个协议已经仔细修改,以适应初级大鼠海马神经元文化的要求。 它也可以应用于更一般的神经元文化目的在微流体。

材料和试剂

  1. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  2. HBSS(Hyclone,目录号:SH30268)
  3. 硼酸(Sigma-Aldrich,目录号:B0252)
  4. 硼砂(Sigma-Aldrich,目录号:B9876)
  5. 半胱氨酸(Sigma-Aldrich,目录号:C7352)
  6. EtOH
  7. 神经基础培养基(NBM)
  8. Glutamax
  9. B27
  10. PenStrep
  11. 庆大霉素
  12. 谷氨酸
  13. FBS
  14. 层粘连蛋白
  15. 台盼蓝
  16. Na 2 4
  17. K 2 SO 4
  18. MgCl 2
  19. CaCl <2>
  20. 葡萄糖
  21. 酚红
  22. EDTA
  23. 0.1M硼酸盐缓冲液(pH 8.5)(参见配方)
  24. 解剖介质(DM)(参见配方)
  25. 木瓜蛋白酶激活缓冲液(参见配方)
  26. 木瓜蛋白酶(100mg)(Worthington Biochemical Corp,目录号:LS003119)(参见Recipes)
  27. DNase(F.Hoffmann-La Roche,目录号:10104159001)(参见Recipes)
  28. 木瓜蛋白酶消化培养基(参见配方)
  29. 解离介质(参见配方)
  30. 用酚红电镀介质(参见配方)
  31. 不含酚红的生长培养基(参见配方)

设备

  1. PDMS设备
  2. 显微镜
  3. 血细胞计数器
  4. TC机罩
  5. 孵化器
  6. -80°C冰箱
  7. 37°C水浴
  8. 6 cm细胞培养皿
  9. 等离子焊接机(型号PDC-32G, http://www.harrickplasma.com/

程序

  1. 盖玻片和腔室准备
    第1天:
    1. PDMS装置如Park等人(2006)中所述制造。
    第2天:
    1. 康宁No.1或者1.5号眼镜都适合我们的显微镜(24 mm x 40 mm)。 或者,Carolina coverglasses 24 mm x 50 mm。
      1. 在高压灭菌的ddH 2 O中对盖玻片进行超声处理60分钟。
      2. 在70%EtOH中再次超声处理30分钟
      3. 在100%EtOH中超声处理30分钟
      4. 在TC罩中用高压灭菌的ddH 2 O洗涤,在罩中将盖玻片空气干燥过夜。
    第3天:
    1. 使用等离子体粘合机组装盖玻片和PDMS装置
    2. 用力按压角落,但只轻轻地触摸中间通道。 用PDL(> 150Kmol wt,在硼砂硼酸缓冲液中制备)涂布室:
      1. 将0.5mg/ml PDL加入到装置一侧的孔中,并允许其流入连接孔(留下未填充的另一侧)。
      2. 允许PDL流过设备约10分钟,然后向孔中添加更多的PDL以填充它们。
      3. 将含有PDL的装置置于37℃的培养箱中至少30分钟,使PDL填充微型槽。
      4. 用PDL填充腔室另一侧的孔和主要通道,并将腔室在37℃下孵育至少4小时,优选过夜。
    第4天:
    1. 处理后吸出多余的PDL,但要小心,不要从设备中吸出所有的液体。
      1. 将高压灭菌的ddH 2 O加到装置两侧的每个孔中,并通过毛细管作用流到另一个孔。
      2. 吸出水(再次小心不要完全从装置中除去所有液体),然后向每个连接的孔中的另一个中再加入150μl高压灭菌的ddH 2 O 2并允许其流过对应井。
      3. 重复该快速洗涤两次,然后将含有ddH 2 O的装置在37℃的培养箱中放置1小时。
      4. 然后,重复洗涤步骤。用ddH 2 O 2每次至少洗涤装置三次,每次一小时,这是总量。
      5. 将具有必需因子的NBM(glutamax,B27,PenStrep)添加到顶部孔中。使其流过,在37℃孵育过夜。
      6. 准备40毫升电镀介质:
        800μlB27 +30μg/ml庆大霉素+400μlglutamax +25μM谷氨酸盐
      7. 在15ml管中预热10ml解离培养基:Neurobasal + 5%FBS +庆大霉素。
      8. 预热10 ml普通neurobasal介质为木瓜蛋白酶
    第5天:
    1. 向每个孔中加入层粘连蛋白(20μg/ml)并在37℃下孵育至少30分钟:用层粘连蛋白包被室:从-80℃冰箱中取出20μl等分试样并立即放在冰上,等待直到 它完全溶解在冰上,然后加入冷的neurobasal媒体到1.8毫升。
      1. 打开紫外线灯,紫外线消毒一个6厘米的细胞培养皿15分钟。
      2. 在该6cm细胞培养皿中,37℃培养箱中预热14ml电镀培养基
      3. 在电镀细胞前1小时将层粘连蛋白溶液替换为电镀培养基

  2. 海马神经元的解剖
    1. 在37℃下用750μl木瓜蛋白酶缓冲液活化木瓜蛋白酶10mg等分试样30分钟
    2. 加入预热的neurobasal培养基,过滤除菌,然后添加一个DNase 20微升等分。
    3. 在TC罩中用10ml冷的解剖培养基洗涤组织(洗涤两次)
    4. 在组织通过重力沉降后,完全去除HBSS
    5. 将一等份的DNA酶加入到10ml消化培养基(昨天预热)中
    6. 将一半消化培养基(5ml)在37℃水浴上放置15分钟
    7. 将剩余的一半消化培养基加入管中,并在37℃水浴下再孵育15分钟
    8. 让组织通过重力沉降到底部或短时间旋转
    9. 用5ml木瓜蛋白酶解离培养基重悬浮沉淀,用5ml移液管上下颠倒5次,避免鼓泡培养基。
    10. 让碎片缓慢沉淀,或短暂旋转到100 g上下(600 rpm),将上清液转移到一个新的15毫升管。
    11. 继续研磨未解离的组织5-10次,取上清液,并与前5毫升结合
    12. 取10微升,并与10微升台盼蓝混合
    13. 加载10μl混合物在血细胞计数器,并计数:
      16个小方格中的所有细胞×10 4个细胞/ml(来自12只E18幼崽的典型产量为〜50M)。

  3. Amaxa转染
    1. 在使用前,将82μl核转染液与18μl补充物混合
    2. 准备50微升nucleofector与5微克总DNA(如果使用多于1质粒,混合等摩尔比)。
    3. 在150×g下旋转5百万个(每个转染的1-6百万个)神经元5分钟。 在纺丝期间,转移7毫升预热的电镀培养基到15毫升管,加入350微升FBS,保持在37℃。 用尖头准备1ml移液器。
    4. 旋转后,使用吸液管用200μl移液器吸头取出所有培养基
    5. 将细胞重悬在50μlnucleofector溶液中
    6. 混合50微升细胞与DNA + neucleofector的预混合物(总共100微升)
    7. Zap与程序G-13(更好的Hc)或O-03(更好的Cx)。 将形成死细胞泡沫。
    8. 立即移液在1毫升加热的平板培养基中的FBS和移液管到1.7毫升eppendorf管。 不要拿起死泡沫。 温柔。
    9. 救援后在37℃下孵育10分钟
    10. 在150分钟,5分钟,150 /分钟下旋转细胞。
    11. 用FBS重悬于300μl电镀培养基中
    12. 加载5μl/孔,填充每个微流体的两个上部孔
    13. 让细胞在37℃下粘附10分钟
    14. 使用带有FBS的电镀培养基的两个移液管同时从两端填充孔
    15. 2小时后将介质完全更换为新鲜的电镀介质W/O FBS
    第6天:
    1. 第二天早上用电镀介质再次更换介质。
    2. 每2天更换一半介质。

食谱

  1. 0.1M硼酸盐缓冲液(pH8.5)(硼砂硼酸缓冲液) 1.24g硼酸
    1.9克硼砂 用ddH 2 O 2加到500ml/dm 2 PH应为8.5,无需调整
    过滤灭菌
    储存于4°C。
  2. 解剖介质(DM)(1L)
    82ml 1M Na 2 SO 4 4·h/v 60ml 0.5M K 2 SO 4 4·h/v 5.8ml 1M MgCl 2·6H/v 252μl1M CaCl 2 8ml 2.5M葡萄糖(20mM) 1ml 1M Hepes(pH 7.4)
    2ml 0.5%苯酚红pH用1M NaOH至7.4过滤灭菌并保持在4℃ 或者只是使用HBSS并加入葡萄糖至20mM,HEPES:
    500ml HBSS与Ca 2+ 2 + 和Mg 2 +
    5ml 1.5M D-葡萄糖(过滤除菌)
    5ml 1.0M HEPES pH 7.2
  3. 木瓜蛋白酶激活缓冲液(50ml)
    1.1mM EDTA110μl(0.5M EDTA,pH8.0) 0.067mMβ-巯基乙醇0.25μl(0.0047μlBME/ml缓冲液) 5.5mM半胱氨酸33.25mg
    储存于4°C。
  4. Papain
    等分到10毫克/1.7毫升埃彭道夫管,存储冻干粉在4℃
    使用前加入750μl木瓜蛋白酶缓冲液
    在37°C下活化至200 U/ml(10 x) - 30分钟(暂时摇动试管一次)
  5. DNase
    1000U /μl储液,20μl等分试样(20,000U /管,用于10ml)
  6. 木瓜蛋白酶消化培养基(每十只动物)
    添加750微升激活的木瓜蛋白酶到纯神经基质培养基
    过滤灭菌
    添加DNase:2 U /μl  DNase
  7. 解离培养基(10ml神经鼻)
    庆大霉素40μg/ml
    FBS 500μl
  8. 用酚红(在20ml神经鼻)中用血清培养培养基
    B27400μl(Gem21,NS21同样)
    庆大霉素40μg/ml(10mg/ml原液,取80μl/20ml培养基)
    Glutamax 200μl
    谷氨酸25μM(100mM储液,4,000x,5μl/20ml)(使用12.5μM终浓度可能更好:2.5〜3μl/20ml)
    FBS 200μl
  9. 不含酚红的生长培养基(在20ml神经鼻中)
    B27400μl(Gem21,NS21同样)
    Glutamax 200μl

致谢

备选参考方案可以在与AXIS TM Axon隔离装置相关的Millipore网站上找到。 解剖方案从在迈克尔林博士的实验室,儿科和生物工程系,美国斯坦福大学开发的协议修改。

参考文献

  1. 可以从 http://www.xonamicrofluidics.com/about.html 请求详细的参考协议。
  2. Park,J.W.,Vahidi,B.,Taylor,A.M.,Rhee,S.W.and Jeon,N.L。(2006)。 神经科学研究的微流体培养平台 Nat Protoc 1 (4):2128-2136。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Geng, Y. (2012). Hippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers. Bio-protocol 2(14): e235. DOI: 10.21769/BioProtoc.235.
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