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Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures
采用乳酸脱氢酶动力测定法检测原代神经元细胞培养物中的细胞损伤   

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Abstract

The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell cultures serve as a practicable tool for quantification of cellular injury (Koh and Choi, 1987; Bruer et al., 1997). Lactate dehydrogenase (LDH) is such a ubiquitously expressed cytosolic enzyme, which is very stable due to a very long protein half-life (Hsieh and Blumenthal, 1956; Koh and Cotman, 1992; Koh et al., 1995).

Keywords: LDH assay(LDH测定), Primary neurons(原代神经元), Cell damage(细胞损伤), LDH release(LDH释放), Cell culture(细胞培养), Cell death(细胞死亡), Survival(生存), Oxygen-glucose deprivation(缺氧缺糖), Glutamate toxicity(谷氨酸盐毒性)

Background

LDH catalyzes the formation of lactate and Nicotinamidadenindinucleotid (NAD+) from pyruvate and reduced Nicotinamidadenindinucleotid (NADH) in a reversible biochemical reaction. NADH has an absorption on the wavelength of 340 nm. The basis of this kinetic LDH activity assay is the decrease of optical density at the specific wave length caused by a decrease of NADH. The amount of LDH in the supernatant is calculated using a standard enzyme solution with known LDH activity. Different cell densities or metabolic activation rates might be confounders; therefore normalization of LDH activity is recommended. This is achieved by assessment of LDH activity after outer cell membrane lysis that does not block LDH activity itself (‘full kill’ with 0.5% Triton-X). Finally, percentage of absolute LDH activity from LDH activity by full kill indicates the rate of damaged or dead cells in the cell culture well.

Materials and Reagents

  1. 96 well plate, flat bottom (SARSTEDT, catalog number: 82.1581 )
  2. Pipette tips
  3. Neuronal cell cultures which was treated by noxes (e.g., oxygen and glucose deprivation, glutamate or other toxins) (Koh and Choi, 1987; Bruer et al., 1997; Harms et al., 2001; Ruscher et al., 2002; Harms et al., 2004; Meisel et al., 2006; Harms et al., 2007; Datwyler et al., 2011; Schweizer et al., 2015; Donath et al., 2016)
    Note: This protocol works for primary neuronal cultures that were derived from various regions of the brain including septum, hippocampus, striatum, spinal cord, cortex, cerebellum or raphe nuclei and that were seeded in various densities or with various media formulations or coatings of the plates. However, we achieve best results with cultures that are at least cultivated for more than 7 days in vitro (DIV) and that were seeded in a reasonable density as illustrated below (Figures 1 and 2). These cultures contain approximately 10% glial cells and this does not impact on the possibility to analyze LDH release in the supernatant medium.


    Figure 1. Primary cortical neuronal cultures derived from mouse E15 embryos after 7 days in vitro (DIV). Scale bar = 50 µm.


    Figure 2. Primary cortical neuronal cultures that were treated with 50 µM glutamate for 24 h on DIV 8. Scale bar = 50 µm.

  4. Triton X-100 solution (Sigma-Aldrich, catalog number: 93443 )
  5. Potassium phosphate monobasic (KH2PO4) (MW 136.1) (Sigma-Aldrich, catalog number: P5655 )
  6. Dibasic potassium phosphate (K2HPO4; MW 174.2) or potassium phosphate trihydrate (K2HPO4·3H2O; MW 228.2) (Sigma-Aldrich catalog number: P5504 )
  7. Distilled water
  8. Na-pyruvate (MW 110) (Sigma-Aldrich, catalog number: P2256 )
  9. LDH standard (TruCal U) (DiaDys Diagnostic Systems, catalog number: 5 9100 99 10 063 )
  10. -NADH (MW 709.4, reduced form) (Sigma-Aldrich, catalog number: N8129 )
  11. 10x LDH-buffer (see Recipes)
  12. 1x LDH-buffer (see Recipes)
  13. LDH-standard (see Recipes)
  14. Na-pyruvate-solution (see Recipes)
  15. -NADH-solution (see Recipes)

Equipment

  1. Pipette
  2. CO2-incubator for cell cultures
  3. Multi-pipette (Eppendorf, model: Multipette® plus )
  4. Spectrophotometer for 96 well plate that can measure 340 nm and kinetic measurement (e.g., Dynex Technologies, model: MRX Microplate Reader )

Procedure

  1. Preparation of solutions (see Recipes)
  2. Measurement
    1. Warm up NADH and an appropriate aliquot of Na-pyruvate-solution (for one 96 well plate you need about 2.5 ml Na-pyruvate-solution) to room temperature or max. 37 °C, thaw appropriate amount of LDH-standard aliquots.
    2. Pipette 50 µl cell culture medium (probe) in each well. A duplicate measurement of each cell culture well is recommend.
    3. Pipette 25 µl LDH standard in 2 wells per plate. It is very important that you perform a standard measurement on each 96 well plate.
    4. Add 200 µl NADH solution to each well and remove bubbles if necessary. The final concentration of NADH in the assay is 153.8 µM.
    5. Add 25 µl Na-pyruvate solution to each well. The enzymatic reaction starts promptly. The final concentration of Na-pyruvate in the assay is 2.063 mM. Do not mix samples with pipets. Mixing should be initiated by using automated shaking by the plate reader before and in between of each measurement cycle. The reaction is within the linear range, if excess NADH is available for the enzymatic reaction. This lasts about 7-10 min. Ideally, pipetting of pyruvate and measurement of all cycles should be accomplished within this period. Do not start the reaction in more than one plate at once. It might be necessary to dilute the sample as specified in Figure 3.


      Figure 3. Representative graph of a measurement. The blue line indicates an undiluted and the red line a diluted (F1:4, red) kinematic curve. Note that the undiluted sample shows a non-linear decrease in absorption at 340 nm.

    6. Immediately start measurement of absorption with following parameters:
      1. Wavelength: 340 nm.
      2. Measurement cycles: 10.
      3. Cycle time: 30 sec.
      4. Shaking time: 5 sec.
      5. Temperature: 37 °C (see also Notes).
  3. Total lysis
    1. Add Triton X-100 in the rest of cell culture medium, the final concentration should be 0.5%.
    2. Incubate on 37 °C for 20 min. It is possible to incubate a longer time until to 4-6 h or overnight. This is not critical as long as you treat all groups equally.
    3. Repeat steps 2b-2f from paragraph 2 (Measurement).

Data analysis

  1. Calculate the decrease of absorption for each well in mAbs/min. In our hands the value is about 10-50 mAbs/min.
    Note: Check, if the decrease of absorption is linear over the whole time. Otherwise reduce the volume of probe or dilute them.
  2. The decrease of absorption in LDH standard equates an LDH activity about 500 U/l. The LDH activity in standard is a little bit different in different batches. Calculate according to manufactures specifications.
  3. Calculate the LDH activity for your probes using a rule of proportion.
    Example:
    1. Decrease of absorption in probe: 15 mAbs/min.
    2. Decrease of absorption in LDH standard: 43 mAbs/min.
    3. LDH activity in standard 466 U/l.
    Equation: (15 mAbs/min x 466 U/l/43 U/l) = 162.5 U/l
  4. Adjust the LDH activity for probe on the relation of probe volume and LDH standard volume.
  5. Finally, calculate LDH activity as a percentage of LDH activity in the same cell culture sample after assessing LDH activity after total LDH release (‘full kill’).

Notes

  1. The reaction rate is dependent on the temperature. In case of high LDH activity in your probe and an additional high temperature an exhaustion of -NADH is possible and leads to a non-linear decrease of absorption.
  2. Assessment of total LDH release after cell lysis (‘full kill’) is recommend for each cell culture well. There are some treatments, which lead to an increase of protein synthesis in cells, so that the amount of total LDH in the cells is increased as well. Normalization helps for better correlation as an indirect measure of cell death in such cases.
  3. It is possible to store samples of supernatants at 4 °C if samples are sealed to avoid evaporation for two to three days with a minimal loss of LDH activity. Do not freeze samples.

Recipes

  1. 10x LDH-buffer
    45.3 g KH2PO4
    116.1 g K2HPO4 or 152 g K2HPO4·3H2O
    Dissolve in about 800 ml distilled water, adjust pH to 7.4 and fill up to 1,000 ml with distilled water
    Store at 4 °C, The solution is stable for 12 month
  2. 1x LDH-buffer
    Dilute 10x LDH buffer with distilled water 1:10
    Store at 4 °C, long-term storage up to 12 month is possible
  3. LDH-standard
    Dissolve lysate of enzyme according to manufactures protocol, prepare aliquots of 105 µl (that’s for measurement of two plates) and store at -20 °C. Avoid freeze and thaw cycles
  4. Na-pyruvate-solution
    Dissolve 1.25 g Na-pyruvate in 500 ml 1x LDH-buffer, results in 22.7 mM Na-pyruvate-solution
    Store at 4 °C. The solution is stable for 18 month
  5. -NADH-solution
    Dissolve 3 mg -NADH in 20 ml 1x LDH-buffer, results in 211.4 µM solution
    Note: 20 ml is the amount for one 96 well plate. This solution is stable only for 2 days at 4 °C.

Acknowledgments

This work was supported by the German Research Foundation (SFB TRR43 and HA5741/1-2 to Christoph Harms), the Federal Ministry of Education and Research (01 EO 08 01) for funding of the Center for Stroke Research Berlin (project ‘SUMO and stroke’ to Christoph Harms), and Berlin Institute of Health, TRG7, TP1 to Christoph Harms.

References

  1. Bruer, U., Weih, M. K., Isaev, N. K., Meisel, A., Ruscher, K., Bergk, A., Trendelenburg, G., Wiegand, F., Victorov, I. V. and Dirnagl, U. (1997). Induction of tolerance in rat cortical neurons: hypoxic preconditioning. FEBS Lett 414(1): 117-121.
  2. Datwyler, A. L., Lattig-Tunnemann, G., Yang, W., Paschen, W., Lee, S. L., Dirnagl, U., Endres, M. and Harms, C. (2011). SUMO2/3 conjugation is an endogenous neuroprotective mechanism. J Cereb Blood Flow Metab 31(11): 2152-2159.
  3. Donath, S., An, J., Lee, S. L., Gertz, K., Datwyler, A. L., Harms, U., Muller, S., Farr, T. D., Fuchtemeier, M., Lattig-Tunnemann, G., Lips, J., Foddis, M., Mosch, L., Bernard, R., Grittner, U., Balkaya, M., Kronenberg, G., Dirnagl, U., Endres, M. and Harms, C. (2016). Interaction of ARC and Daxx: A novel endogenous target to preserve motor function and cell loss after focal brain ischemia in mice. J Neurosci 36(31): 8132-8148.
  4. Harms, C., Albrecht, K., Harms, U., Seidel, K., Hauck, L., Baldinger, T., Hubner, D., Kronenberg, G., An, J., Ruscher, K., Meisel, A., Dirnagl, U., von Harsdorf, R., Endres, M. and Hortnagl, H. (2007). Phosphatidylinositol 3-Akt-kinase-dependent phosphorylation of p21(Waf1/Cip1) as a novel mechanism of neuroprotection by glucocorticoids. J Neurosci 27(17): 4562-71.
  5. Harms, C., Bosel, J., Lautenschlager, M., Harms, U., Braun, J. S., Hortnagl, H., Dirnagl, U., Kwiatkowski, D. J., Fink, K. and Endres, M. (2004). Neuronal gelsolin prevents apoptosis by enhancing actin depolymerization. Mol Cell Neurosci 25(1): 69-82.
  6. Harms, C., Lautenschlager, M., Bergk, A., Katchanov, J., Freyer, D., Kapinya, K., Herwig, U., Megow, D., Dirnagl, U., Weber, J. R. and Hortnagl, H. (2001). Differential mechanisms of neuroprotection by 17 β-estradiol in apoptotic versus necrotic neurodegeneration. J Neurosci 21(8): 2600-2609.
  7. Hsieh, K. M. and Blumenthal, H. T. (1956). Serum lactic dehydrogenase levels in various disease states. Proc Soc Exp Biol Med 91(4): 626-630.
  8. Koh, J. Y. and Choi, D. W. (1987). Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods 20(1): 83-90.
  9. Koh, J. Y. and Cotman, C. W. (1992). Programmed cell death: its possible contribution to neurotoxicity mediated by calcium channel antagonists. Brain Res 587(2): 233-240.
  10. Koh, J. Y., Gwag, B. J., Lobner, D. and Choi, D. W. (1995). Potentiated necrosis of cultured cortical neurons by neurotrophins. Science 268(5210): 573-575.
  11. Meisel, A., Harms, C., Yildirim, F., Bosel, J., Kronenberg, G., Harms, U., Fink, K. B. and Endres, M. (2006). Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation. J Neurochem 98(4): 1019-1031.
  12. Ruscher, K., Freyer, D., Karsch, M., Isaev, N., Megow, D., Sawitzki, B., Priller, J., Dirnagl, U. and Meisel, A. (2002). Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model. J Neurosci 22(23): 10291-301.
  13. Schweizer, S., Harms, C., Lerch, H., Flynn, J., Hecht, J., Yildirim, F., Meisel, A. and Marschenz, S. (2015). Inhibition of histone methyltransferases SUV39H1 and G9a leads to neuroprotection in an in vitro model of cerebral ischemia. J Cereb Blood Flow Metab 35(10): 1640-1647.

简介

神经生物学领域中许多急性或慢性退行性疾病的体外模型的目的是评估神经元细胞的存活或损伤。细胞损伤与外细胞膜完整性的丧失和细胞质细胞蛋白的泄漏有关。因此,细胞培养上清液中细胞质酶的活性测定作为细胞损伤定量的实用工具(Koh和Choi,1987; Bruer等,1997)。乳酸脱氢酶(LDH)是一种无处不在表达的细胞溶质酶,由于蛋白质的半衰期很长,其稳定性很高(Hsieh和Blumenthal,1956; Koh和Cotman,1992; Koh等人)。 ,1995)。

背景 LDH在可逆的生物化学反应中催化丙酮酸和还原的烟酰胺偶氮二核苷酸(NADH)的乳酸盐和烟酰胺氨基茚三核苷酸(NAD +)的形成。 NADH在340nm的波长上具有吸收。这种动力学LDH活性测定的基础是由NADH降低引起的特定波长处的光密度降低。使用具有已知LDH活性的标准酶溶液计算上清液中LDH的量。不同的细胞密度或代谢活化率可能是混杂的;因此推荐LDH活性的正常化。这是通过评估外部细胞膜裂解后不抑制LDH活性的LDH活性(用0.5%Triton-X“完全杀死”)来实现的。最后,通过完全杀死的LDH活性的绝对LDH活性百分比表示细胞培养物中损伤或死细胞的发生率。

关键字:LDH测定, 原代神经元, 细胞损伤, LDH释放, 细胞培养, 细胞死亡, 生存, 缺氧缺糖, 谷氨酸盐毒性

材料和试剂

  1. 96孔板,平底(SARSTEDT,目录号:82.1581)
  2. 移液器提示
  3. 由noxes(例如,氧和葡萄糖剥夺,谷氨酸或其他毒素)处理的神经元细胞培养物(Koh和Choi,1987; Bruer等人,1997; Harms等人,2001; Ruscher等人,2002; Harms等人,2004; Meisel等人[ ,2006; Harms等人,2007; Datwyler等人,2011; Schweizer等人,2015; Donath em>等,,2016)
    注意:该协议适用于源于大脑各个区域的主要神经元培养物,包括隔膜,海马,纹状体,脊髓,皮质,小脑或裂核,并以各种密度或各种培养基制剂或板的涂层。然而,我们通过体外培养超过7天的培养物(DIV)获得最佳结果,并以如下所示的合理密度接种(图1和图2)。这些培养物含有约10%的神经胶质细胞,这不会影响分析上清液中LDH释放的可能性。


    图1.来自小鼠E15胚胎的原代皮层神经元培养物在体外7天后(DIV)。比例尺=50μm。


    图2.在DIV 8上用50μM谷氨酸盐处理24小时的原代皮层神经元培养物。比例尺=50μm。

  4. Triton X-100溶液(Sigma-Aldrich,目录号:93443)
  5. 磷酸二氢钾(KH 2 PO 4)(MW 136.1)(Sigma-Aldrich,目录号:P5655)
  6. 磷酸二氢钾(K 2/2 HPO 4 MW; MW 174.2)或磷酸三钾水合物(K 2 HPO 4) 3H 2 O; MW 228.2)(Sigma-Aldrich目录号:P5504)
  7. 蒸馏水
  8. 丙酮酸钠(MW110)(Sigma-Aldrich,目录号:P2256)
  9. LDH标准(TruCal U)(DiaDys Diagnostic Systems,目录号:5 9100 99 10 063)
  10. -NADH(MW 709.4,还原形式)(Sigma-Aldrich,目录号:N8129)
  11. 10x LDH缓冲液(见配方)
  12. 1x LDH缓冲(见配方)
  13. LDH标准(见配方)
  14. 丙酮酸钠溶液(参见食谱)
  15. -NADH-溶液(参见食谱)

设备

  1. 移液器
  2. CO 2 - 细胞培养物的培养基
  3. 多吸管(Eppendorf,型号:Multipette ® plus)
  4. 用于96孔板的分光光度计,其可以测量340nm并进行动力学测量(例如,Dynex Technologies,型号:MRX Microplate Reader)

程序

  1. 解决方案的准备(见配方)
  2. 测量
    1. 加热NADH和适量的丙酮酸钠溶液(对于一个96孔板,您需要约2.5毫升的丙酮酸钠溶液)至室温或最大。 37℃,解冻适量的LDH标准等分试样。
    2. 移取每孔50μl细胞培养液(探针)。推荐每个细胞培养物的重复测量。
    3. 在每个板2个孔中吸取25μlLDH标准液。在每个96孔板上执行标准测量非常重要。
    4. 向每个孔中加入200μlNADH溶液,并在必要时清除气泡。测定中NADH的最终浓度为153.8μM
    5. 向每个孔中加入25μl的丙酮酸钠溶液。酶反应迅速开始。测定中的丙酮酸钠的最终浓度为2.063mM。不要将样品与移液管混合。应通过在每个测量周期之前和之间的平板阅读器自动振荡来启动混合。如果过量的NADH可用于酶反应,则反应在线性范围内。这持续约7-10分钟。理想情况下,丙酮酸的吸取和所有循环的测量应在此期间完成。不要一次在多于一个的板上开始反应。可能需要稀释样品,如图3所示

      图3.测量的代表性图。蓝线表示未稀释,红线表示稀释(F1:4,红色)运动曲线。请注意,未稀释的样品在340 nm处显示吸收的非线性降低。

    6. 立即用以下参数开始吸收测量:
      1. 波长:340 nm
      2. 测量周期:10.
      3. 周期时间:30秒。
      4. 震动时间:5秒
      5. 温度:37°C(另见注)。
  3. 总溶解
    1. 在细胞培养基的其余部分添加Triton X-100,最终浓度应为0.5%
    2. 在37℃下孵育20分钟。可以孵育更长时间直到4-6小时或过夜。只要你平等对待所有团体,这并不重要
    3. 重复第2段(测量)中的步骤2b-2f。

数据分析

  1. 计算每个孔的吸收减少量,单位为mAbs / min。在我们手中,值约为10-50 mAbs / min。
    注意:检查,如果吸收的减少在整个时间内是线性的。否则减小探头的体积或稀释。
  2. LDH标准吸收的降低等于LDH活性约为500 U / L。标准中的LDH活动在不同批次中有所不同。根据制造商规格计算。
  3. 使用比例规则计算探针的LDH活性。
    示例:
    1. 探头吸收减少:15 mAbs / min。
    2. LDH标准吸收减少:43 mAbs / min
    3. LDH活性标准为466 U / l。
    方程式:(15 mAbs / min x 466 U / l / 43 U / l)= 162.5 U / l
  4. 根据探头体积和LDH标准体积的关系调整探针的LDH活性。
  5. 最后,在总LDH释放(“完全杀死”)后评估LDH活性后,计算LDH活性在同一细胞培养物样品中LDH活性的百分比。

笔记

  1. 反应速率取决于温度。如果您的探针具有较高的LDH活性,并且额外的高温可能会导致-NADH的耗尽,并导致吸收的非线性降低。
  2. 细胞裂解后的总LDH释放量(“完全杀死”)的评估推荐用于每种细胞培养。有一些治疗方法,导致细胞中蛋白质合成的增加,使得细胞中总LDH的量也增加。在这种情况下,归一化有助于更好地作为间接测量细胞死亡的相关性
  3. 如果样品被密封,可以将样品的上清液储存在4℃,以避免蒸发2至3天,同时LDH活性降低最小。不要冻结样品。

食谱

  1. 10x LDH缓冲区
    45.3g KH 2 PO 4
    116.1g K 2 HPO 4或152g K 2 HPO 4·3H 2 O
    溶解在约800毫升蒸馏水中,调节pH至7.4,并用蒸馏水填充至1000毫升 存储在4°C,解决方案稳定12个月
  2. 1x LDH缓冲区
    用蒸馏水稀释10倍LDH缓冲液1:10
    存储在4°C,长期存储长达12个月可能
  3. LDH标准
    根据制造方案溶解酶的裂解物,制备105μl的等分试样(用于测量两个板)并储存在-20℃。避免冻融循环
  4. 丙酮酸钠溶液
    将1.25 g的丙酮酸钠溶解于500 ml 1x LDH缓冲液中,得到22.7 mM的丙酮酸钠溶液, 储存于4°C。该解决方案稳定18个月
  5. -NADH解决方案
    将3毫克-NADH溶于20毫升1x LDH缓冲液中,得到211.4微克溶液 注意:20毫升是一个96孔板的量。该解决方案仅在4°C下稳定2天。

致谢

这项工作由德国研究基金会(SFB TRR43和HA5741 / 1-2至Christoph Harms),联邦教育和研究部(01 EO 08 01)支持,为柏林中风研究中心提供资金(项目“SUMO和克里斯托弗·哈姆斯(Christoph Harms),柏林健康研究所TRG7,TP1,Christoph Harms。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Freyer, D. and Harms, C. (2017). Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures. Bio-protocol 7(11): e2308. DOI: 10.21769/BioProtoc.2308.
  2. Donath, S., An, J., Lee, S. L., Gertz, K., Datwyler, A. L., Harms, U., Muller, S., Farr, T. D., Fuchtemeier, M., Lattig-Tunnemann, G., Lips, J., Foddis, M., Mosch, L., Bernard, R., Grittner, U., Balkaya, M., Kronenberg, G., Dirnagl, U., Endres, M. and Harms, C. (2016). Interaction of ARC and Daxx: A novel endogenous target to preserve motor function and cell loss after focal brain ischemia in mice. J Neurosci 36(31): 8132-8148.
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