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Manganese Cytotoxicity Assay on Hippocampal Neuronal Cell Culture
海马神经元细胞培养的锰细胞毒性分析

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Abstract

Compared to an in vivo experiment, neuronal cell cultures are immediately accessible to observation and manipulation. In this protocol, we describe a technique to evaluate the cytotoxicity of a metal, manganese (Mn2+), on hippocampal neuronal cell cultures. Interestingly, this protocol is easily adaptable to any type of primary culture (e.g., cortical neurons) and any type of toxic compound (e.g., chemical product).
This protocol is similar to "Neuron-enriched Cultures (Method 2)" protocol (Gao, 2011).

Keywords: MRI(MRI), MEMRI(这种方法), Axonal transport(轴突运输), Manganese enhanced MRI(锰增强磁共振成像)

Materials and Reagents

  1. Pregnant mouse (embryonic day: E18.5)
  2. Borax (Sigma-Aldrich, catalog number: 71997 )
  3. Boric acid (Sigma-Aldrich, catalog number: B6768 )
  4. 50x B27 supplement (Life Technologies, InvitrogenTM, catalog number: 17504-044 )
  5. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  6. Dulbeccos modified Eagle medium (DMEM) (Life Technologies, InvitrogenTM, catalog number: 31966-021)
  7. Fetal bovine serum (FBS) (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
  8. 100x glutamax (Life Technologies, InvitrogenTM, catalog number: 35050-038)
  9. 10x HBSS (Life Technologies, InvitrogenTM, catalog number: 14185-052 )
  10. 1 M MnCl2 (Sigma-Aldrich, catalog number: M1787 )
  11. Neurobasal medium (Life Technologies, InvitrogenTM, catalog number: 21103-049 )
  12. Penicillin-streptomycin (PS) (10,000 U/ml) (Life Technologies, InvitrogenTM, catalog number: 15140-122 )
  13. Dulbecco’s phosphate buffer saline (DPBS) with Ca2+ and Mg2+ (Sigma-Aldrich, catalog number: D8662 )
  14. Poly-L-Lysine hydrobromide (Sigma-Aldrich, catalog number: P2636 )
  15. Sterile water
  16. Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M2128 )
  17. 10x trypsine (Life Technologies, InvitrogenTM, catalog number: 15090-046 )
  18. Borate buffer (see Recipes)
  19. 1 mg/ml poly-L-lysine (see Recipes)
  20. First culture medium (see Recipes)
  21. 1x HBSS (see Recipes)
  22. Second culture medium (see Recipes)
  23. MnCl2 (0, 20, 50, 100 and 150 µM) (see Recipes)
  24. 0.5 mg/ml MTT (see Recipes)

Equipment

  1. One sterile curved plier and one sterile sharp plier (Figure 1)
  2. One bag of sterile plastic Petri dishes (100 mm diameter)
  3. 24 wells plate (sterile)
  4. 96 wells plate (non sterile)
  5. Syringe filter
  6. Pipette P1000 with a P1000 cone and a P200 cone (sterile)
  7. Neubauer or Malassez cell counting chamber
  8. Eppendorf tubes
  9. Cell scraper
  10. Tissue culture hood
  11. Optic microscope inside the hood
  12. Water bath at 37 °C
  13. Incubator at 37 °C (humidified atmosphere with 5% CO2)
  14. Microplate reader (Pherastar plus) (BMG LABTECH)


    Figure 1. Pliers for the hippocampi dissection

Procedure

Note: All steps are performed under a cell culture hood, using aseptic techniques.

  1. Well plate preparation (Day 1-2)
    Day 1
    1. Place 300 µl of poly-L-lysine 1 mg/ml in all wells of the 24 well plate. Poly-L-lysine is an attachment factor, which improves cell adherence.
    2. Incubate over night at room temperature.
    Day 2, before part B
    1. Remove the poly-L-lysine.
    2. Rinse 2 times (quickly), then rinse for 1 h and finish with 2 rinses (quickly) with sterile water.
    3. Add 1.5 ml of the first culture medium per well.
    4. Place the plate in the incubator until step B9.

  2. Hippocampal neurons preparation (Day 2)
    1. Remove hippocampi from the embryo brain in 1x HBSS using the pliers and the microscope, Figure 1 [see Figure 1A-F in the article of Fuller and Dailey (Fuller and Dailey, 2007)].
    2. Place hippocampi in a tube with 4.5 ml of 1x HBSS.
    3. Add 500 µl of 10x trypsin.
    4. Incubate in a water bath at 37 °C (98.6 °F) for 15 min.
    5. Rinse 3 times with 1x HBSS leaving the tissue at the bottom of the tube.
    6. Add 500 µl of the first culture medium.
    7. Using a P1000 pipette with a P1000 cone and a P200 cone, perform the mechanical dissociation (Figure 2) by sucking and spitting several times the cone content with the pipette. During this step, be careful not to create air bubbles in the medium (air bubbles are toxic for neurons).
    8. Wait a few minutes until the aggregate settles to the bottom of the tube.
    9. Transfer supernatant containing dispersed cells to a sterile Eppendorf tube. Leave ~25 µl of culture medium containing aggregate.
    10. With a Neubauer or Malassez cell counting chamber, count the number of cells and adjust to a number of 2.2 x 105 cells per well. Usually, there are around 1 x 106 hippocampal cells per embryo, so one embryo can be used to fill 5 wells approximately.
    11. Place in the incubator for 2 h.
    12. Replace the first culture medium by the second culture medium (1.5 ml per well).
    13. Place in the incubator for 2 days (until part C).


      Figure 2. Mechanical dissociation using a P1000 pipette with a P1000 cone and a P200 cone

  3. Addition of MnCl2 in the medium culture (Day 4)
    In this protocol, 5 dilutions of MnCl2 are studied: 0, 20, 50, 100 and 150 µM.
    Replace the culture medium by the Mn solutions at different concentrations and place the plate in the incubator for 24 h.

  4. MTT assay (Day 5)
    MTT assay is a fast method for the enumeration of living cells. The tetrazolium salt (yellow) is reduced into formazan by the mitochondrial succinate dehydrogenase of living cells. This reaction creates a purple precipitate.
    1. Prepare fresh MTT solutions 5 mg/ml.
    2. Remove the culture medium and add 500 µl of MTT 0.5 mg/ml diluted in the second medium culture.
    3. Incubate the plate for 2 h at 37 °C.
    4. Induce cell lysis by replacing MTT solution by 500 µl of pure DMSO. Cells will liberate the purple precipitate formed by mitochondria of living cells.
    5. Using a P1000 pipette, help cell lysis by sucking and spitting several times with the pipette.
    6. Detach the last cells still attached to the plate with a cell scraper.
    7. Transfer the lysate into an Eppendorf tube.
    8. Take 200 µl of each lysate and place them in a 96 well plate.
    9. Fill one well with 200 µl of pure DMSO for reference. The optic density of this reference-which contains 0 cells-will be subtracted from the other optic densities.
    10. Using the spectrophotometer, read the optic density of each well filled with the lysate using a wavelength = 519 nm. An absorbance inferior to 1 is necessary to respect the law of Beer Lambert. If the absorbance is above 1, the lysate must be diluted in pure DMSO. The typical absorbance for a culture of 2.2 x 105 neurons at Day 5 is 0.93 ± 0.09. The well without Mn is considered to contain 100% of living cells. You can then compute the fraction of living cells in the other wells from the ratio between the optic density in the wells and the optic density in the well without Mn.

Recipes

  1. Borate buffer
    Mix the boric acid 3.1 g/ml with borax 4.75 g/ml in distilled water
    For a final volume of 50 ml of distilled water, weigh 155 g of boric acid and 237.5 g of borax Adjust the pH to 8.5 with 1 M NaOH
    Sterilized the solution by filtration
  2. 1 mg/ml poly-L-lysine
    1. Prepare a stock solution of poly-L-lysine 10 mg/ml diluted in borate buffer.
    2. For a final volume of 10 ml, weigh 100 mg of poly-L-lysine in 10 ml of borate buffer. Place the stock solution at -20 °C.
    3. Use the stock solution to make a final solution of poly-L-lysine at 1 mg/ml diluted in borate buffer. For a final volume of 10 ml, pipette 1 ml of stock solution + 9 ml of borate buffer.
  3. First culture medium
    This first culture medium is composed of 10% FBS, 1% PS diluted in DMEM.
    For a final volume of 40 ml, mix 4 ml of FBS, 0.4 ml of PS and 35.6 ml of DMEM.
  4. 1x HBSS
    10x HBSS is diluted in sterile water.
    For a final volume of 50 ml, mix 5 ml of HBSS 10x with 45 ml of sterile water.
  5. Second culture medium
    The second culture medium is composed of glutamax 1x and B27 d=1/50 diluted in Neurobasal. For a final volume of 40 ml, mix 0.4 ml of glutamax, 0.8 ml of B27 and 38.8 ml of Neurobasal.
  6. MnCl2 (0, 20, 50, 100 and 150 µM)
    1. Prepare a solution of MnCl2 at 1 mM in distilled water (from the solution at 1 M).
    2. For a final volume of 10 ml, mix 10 µl of MnCl2 1 M and 10 ml of distilled water.
    3. Then, prepare the different concentrations of MnCl2 in the second culture medium.
    4. The final volume of each well is 1.5 ml. Calculate the number of wells that you want to use for each concentration. For example, if you use 3 wells per concentration, you need 4.5 ml of MnCl2 solution (3 x 1.5 ml). The solution preparation is described in Table 1 for a final volume of 4.5 ml.

      Table 1. Preparation of MnCl2 solutions at different concentrations. This example is given for 3 wells per concentration and 4 Mn concentrations.
      Concentration of MnCl2 (µM)
      Volume of MnCl2 1 mM (µl)
      Second culture medium (ml)
      0
      0
      4.5
      20
      90
      4.41
      50
      225
      4.28
      100
      450
      4.05
      150
      672
      3.83

  7. 0.5 mg/ml MTT
    1. First, prepare a MTT solution at 5 mg/ml diluted in PBS with Ca2+ and Mg2+. For 1.2 ml of PBS, weigh 6 mg of MTT. Protect the solution from light.
    2. Second, prepare a MTT solution at 0.5 mg/ml in the second culture medium. For a final volume of 12 ml of culture medium, add 1.2 ml of MTT at 5 mg/ml.

Acknowledgments

AD received a stipend from the Région Rhône-Alpes-Cluster HVN.

References

  1. Daoust, A., Saoudi, Y., Brocard, J., Collomb, N., Batandier, C., Bisbal, M., Salome, M., Andrieux, A., Bohic, S. and Barbier, E. L. (2014). Impact of manganese on primary hippocampal neurons from rodents. Hippocampus 24(5): 598-610.
  2. Fuller, L. and Dailey, M. E. (2007). Preparation of rodent hippocampal slice cultures. CSH Protoc 2007: pdb prot4848.
  3. Gao, H. M. (2011). Neuron-enriched cultures (method 2). Bio-protocol Bio101: e150.

简介

与体内实验相比,神经元细胞培养物可立即进行观察和操作。 在该协议中,我们描述了评价金属锰(Mn 2 + )对海马神经元细胞培养物的细胞毒性的技术。 有趣的是,该方案容易适用于任何类型的原代培养物(例如,皮层神经元)和任何类型的有毒化合物(例如,化学产品)。
此协议类似于"神经元富集培养物(方法2)"方案(Gao,2011) 。

关键字:MRI, 这种方法, 轴突运输, 锰增强磁共振成像

材料和试剂

  1. 怀孕老鼠(胚胎日:E18.5)
  2. 硼砂(Sigma-Aldrich,目录号:71997)
  3. 硼酸(Sigma-Aldrich,目录号:B6768)
  4. 50x B27补充物(Life Technologies,Invitrogen TM ,目录号:17504-044)
  5. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  6. Dulbeccos改良的Eagle培养基(DMEM)(Life Technologies,Invitrogen TM ,目录号:31966-021)
  7. 胎牛血清(FBS)(Life Technologies,Invitrogen TM ,目录号:16000-044)
  8. 100x glutamax(Life Technologies,Invitrogen TM ,目录号:35050-038)
  9. 10x HBSS(Life Technologies,Invitrogen TM ,目录号:14185-052)
  10. 1 M MnCl 2(Sigma-Aldrich,目录号:M1787)
  11. Neurobasal培养基(Life Technologies,Invitrogen TM ,目录号:21103-049)
  12. 青霉素 - 链霉素(PS)(10,000U/ml)(Life Technologies,Invitrogen TM,目录号:15140-122)
  13. 具有Ca 2+和Mg 2+的Sigma-Aldrich的Dulbecco's磷酸盐缓冲盐水(DPBS)(Sigma-Aldrich,目录号:D8662)
  14. 聚-L-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P2636)
  15. 无菌水
  16. 噻唑基蓝四唑溴化物(MTT)(Sigma-Aldrich,目录号:M2128)
  17. 10x胰蛋白酶(Life Technologies,Invitrogen TM ,目录号:15090-046)
  18. 硼酸缓冲液(参见配方)
  19. 1mg/ml聚-L-赖氨酸(见Recipes)
  20. 第一种培养基(见配方)
  21. 1x HBSS(请参阅配方)
  22. 第二种培养基(见配方)
  23. MnCl 2(0,20,50,100和150μM)(参见配方)
  24. 0.5 mg/ml MTT(参见配方)

设备

  1. 一个无菌弯曲钳和一个无菌尖钳(图1)
  2. 一袋无菌塑料培养皿(直径100mm)
  3. 24孔板(无菌)
  4. 96孔板(无菌)
  5. 注射器过滤器
  6. 具有P1000锥体和P200锥体(无菌)的移液器P1000
  7. Neubauer或Malassez细胞计数室
  8. Eppendorf管
  9. 细胞刮刀
  10. 组织培养罩
  11. 镜头内的光学显微镜
  12. 37℃水浴
  13. 37℃(具有5%CO 2的湿润气氛)的培养箱中培养
  14. 酶标仪(Pherastar plus)(BMG LABTECH)


    图1.海马解剖用钳子

程序

注意:所有步骤都在细胞培养罩下进行,使用无菌技术。

  1. 孔板制备(第1-2天)
    第1天
    1. 放置300微升聚-L-赖氨酸1毫克/毫升在24孔的所有井 盘子。 聚-L-赖氨酸是一种附着因子,其改善细胞 坚持。
    2. 在室温下孵育过夜。
    第2天,在B部分之前
    1. 删除聚-L-赖氨酸。
    2. 冲洗2次(快速),然后冲洗1小时,用无菌水冲洗2次(快速)。
    3. 每孔加入1.5ml第一培养基。
    4. 将板放在培养箱中,直到步骤B9

  2. 海马神经元制备(第2天)
    1. 删除海马从胚胎脑在1x HBSS使用钳子和 显微镜,图1 [见图1A-F在Fuller和 Dailey(Fuller和Dailey,2007)]。
    2. 将海马放置在带有4.5ml 1×HBSS的管中
    3. 加入500μl10×胰蛋白酶
    4. 在37℃(98.6°F)的水浴中孵育15分钟
    5. 用1×HBSS冲洗3次,使组织在管底部。
    6. 加入500μl的第一培养基。
    7. 使用P1000移液器与P1000锥和P200锥,执行   机械解离(图2)通过吸取和吐出几个 用移液管吸取锥体内容物。 在此步骤中,请小心 不会在介质中产生气泡(气泡有毒性 神经元)。
    8. 等待几分钟,直到骨料沉降到管的底部。
    9. 转移含有分散细胞的上清液至无菌 Eppendorf管。 离开〜25微升含有骨料的培养基。
    10. 使用Neubauer或Malassez细胞计数室,计数数量   的细胞并调整至每孔2.2×10 5个细胞的数目。 通常, 每个胚胎有约1×10 6个海马细胞,因此一个胚胎可以   用于大约填充5口井
    11. 放置在孵化器中2小时。
    12. 用第二培养基替换第一培养基(每孔1.5ml)。
    13. 放置在孵化器中2天(直到C部分)

      图2.使用P1000移液器和P1000锥体和P200锥体的机械分离

  3. 在培养基培养物中添加MnCl 2(第4天)
    在该方案中,研究MnCl 2的5种稀释液:0,20,50,100和150μM。
    用不同浓度的Mn溶液替换培养基,将板置于培养箱中24小时
  4. MTT测定(第5天)
    MTT测定是活细胞计数的快速方法。 通过活细胞的线粒体琥珀酸脱氢酶将四唑鎓盐(黄色)还原成甲。。 该反应产生紫色沉淀。
    1. 准备新鲜的MTT溶液5 mg/ml
    2. 取出培养基,加入稀释在第二培养基中的500μlMTT 0.5mg/ml
    3. 在37℃下孵育平板2小时。
    4. 通过用500μl纯DMSO代替MTT溶液诱导细胞裂解。 细胞将释放由线粒体形成的紫色沉淀 活细胞
    5. 使用P1000移液器,通过吸取和吐痰几次帮助细胞裂解
    6. 用细胞刮刀分离仍附着在板上的最后一个细胞
    7. 将裂解物转移到Eppendorf管中
    8. 取200μl的每个裂解物,并将其放入96孔板
    9. 填充一个井用200微升的纯DMSO作为参考。 光学 将从中减去该参考(其包含0个单元)的密度   其他光密度
    10. 使用分光光度计,读数 使用波长用裂解物填充的每个孔的光密度 = 519nm。 遵守法则需要吸光度低于1 的啤酒。 如果吸光度大于1,则裂解物必须是 稀释在纯DMSO中。 在第5天,2.2×10 5个神经元的培养物的典型吸光度为0.93±0.09。 认为没有Mn的井 含有100%的活细胞。 然后可以计算的分数 活细胞中其他孔的光密度之比   在孔中和在没有Mn的孔中的光密度。

食谱

  1. 硼酸盐缓冲液
    将硼酸3.1g/ml与硼砂4.75g/ml在蒸馏水中混合
    对于最终体积为50ml的蒸馏水,称重155g硼酸和237.5g硼砂。用1M NaOH调节pH至8.5。
    通过过滤灭菌溶液
  2. 1mg/ml聚-L-赖氨酸
    1. 制备稀释在硼酸盐缓冲液中的聚-L-赖氨酸10 mg/ml的储备溶液。
    2. 对于10ml的最终体积,称量100mg的聚-L-赖氨酸在10ml硼酸盐缓冲液中。 将储备液置于-20°C
    3. 使用储备溶液使聚-L-赖氨酸的最终溶液为1   mg/ml稀释在硼酸盐缓冲液中。 对于终体积为10ml,移液管1 ml储备溶液+ 9ml硼酸盐缓冲液。
  3. 第一培养基
    该第一培养基由在DMEM中稀释的10%FBS,1%PS组成 对于40ml的最终体积,混合4ml FBS,0.4ml PS和35.6ml DMEM。
  4. 1x HBSS
    将10x HBSS在无菌水中稀释 对于最终体积为50ml,将5ml HBSS 10x与45ml无菌水混合
  5. 第二培养基
    第二培养基由在Neurobasal中稀释的glutamax 1x和B27 d = 1/50组成。 对于40ml的最终体积,混合0.4ml glutamax,0.8ml B27和38.8ml Neurobasal。
  6. MnCl 2(0,20,50,100和150μM)
    1. 制备1mM的MnCl 2溶液在蒸馏水中(来自1M的溶液)。
    2. 对于最终体积为10ml,混合10μl的MnCl 2 1M和10ml的蒸馏水。
    3. 然后,在第二培养基中制备不同浓度的MnCl 2
    4. 每个孔的最终体积为1.5ml。 计算的数量 你想要用于每个浓度的孔。 例如,如果你 每个浓度使用3个孔,需要4.5ml的MnCl 2溶液(3× 1.5ml)。 溶液制备在表1中描述为最终的 体积为4.5ml
      表1.制备MnCl 2 不同浓度。此示例针对3个孔 浓度和4 Mn浓度
      MnCl 2浓度(μm)
      MnCl 2的体积1mM(μl)
      第二培养基(ml)
      0
      0
      4.5
      20
      90
      4.41
      50
      225
      4.28
      100
      450
      4.05
      150
      672
      3.83

  7. 0.5 mg/ml MTT
    1. 首先,制备用Ca 2+和PBS稀释的PBS中5mg/ml的MTT溶液 Mg 2 + 。 对于1.2ml的PBS,称重6mg的MTT。 保护溶液 光。
    2. 第二,在第二个中制备0.5mg/ml的MTT溶液 培养基。 对于终体积为12ml的培养基,加入1.2 ml的5mg/ml的MTT。

致谢

AD从RégionRhône-Alpes-Cluster HVN获得了奖学金。

参考文献

  1. Daoust,A.,Saoudi,Y.,Brocard,J.,Collomb,N.,Batandier,C.,Bisbal,M.,Salome,M.,Andrieux,A.,Bohic,S。和Barbier, )。 锰对啮齿动物原代海马神经元的影响 海马 24(5):598-610。
  2. Fuller,L。和Dailey,M.E。(2007)。 啮齿类动物海马切片培养物的制备 CSH Protoc 2007 :pdb prot4848。
  3. Gao,H.M。(2011)。 神经元富集培养(方法2) 生物协议 Bio101: e150。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Daoust, A., Saoudi, Y., Brocard, J., Collomb, N., Batandier, C., Bisbal, M., Salomé, M., Andrieux, A., Bohic, S. and Barbier, E. L. (2015). Manganese Cytotoxicity Assay on Hippocampal Neuronal Cell Culture. Bio-protocol 5(1): e1368. DOI: 10.21769/BioProtoc.1368.
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