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Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
Neuro2A细胞和大鼠原代皮层神经元中突变亨廷顿蛋白的分泌   

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本实验方案简略版
The Journal of Neuroscience
Sep 2017

Abstract

Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary cortical neurons by Western blotting. Our protocol is based on reducing the amounts of BSA in the media while maintaining cell viability and secretory potential, and concentrating the media prior to analysis by means of ultrafiltration.

Keywords: Protein secretion (蛋白质分泌), Ultrafiltration (超滤), BSA (BSA)

Background

A number of proteins are secreted from the cell into the extracellular environment via various secretory pathways. These pathways include the conventional secretory pathway following ER-Golgi-plasma membrane route (Lee et al., 2004) and multiple unconventional pathways, such as lysosomal exocytosis, translocation across the plasma membrane and exosome and ectosome release (Zhang and Schekman, 2013). To study these pathways, it is often necessary to analyze proteins secreted from cultured cells into the media. Proteins may be secreted either in free form or in association with vesicular membrane structures such as ectosomes and exosomes (Zhang and Schekman, 2013). Whether these proteins are membrane-associated or not determines the type of approach used for their analysis. To isolate membrane-associated proteins, media is usually subjected to differential centrifugation procedure upon which the protein-containing membranes are sedimented, concentrated and purified from the constituents of the media, which allows for unhampered protein analysis (Momen-Heravi et al., 2013). However, the analysis of free-form proteins is a more challenging task (Chevallet et al., 2007). Two major obstacles are 1) the presence of substantial amounts of bovine serum albumin (BSA) in the serum or other supplements added to most cell culture media, which may mask the protein of interest; and 2) low abundance in the media of at least some of the secreted proteins, including mHtt. This protocol has been developed in order to study secreted mHtt and may be useful for analyzing other low-abundance, free-form proteins in the media.

Protocol optimization
To tackle the issue of excessive BSA, we first monitored cell viability in various media with partial or total reduction of BSA. In the case of Neuro2A cells stably expressing N-terminal 571 amino acids of mHtt with 72 glutamines (Neuro2A-mHtt cells), the optimal medium was Opti-MEM, which not only preserved viability of the cells, but also enhanced the secretion of mutant huntingtin (mHtt) as compared to other media, such as DMEM without supplemented fetal bovine serum (FBS) and HBSS (Figure 1). Moreover, we observed that applying conditioned Opti-MEM (Opti-MEM pre-incubated with naïve Neuro2A cells which do not express mHtt) dramatically enhanced the secretion of mHtt from Neuro2A-mHtt cells, possibly due to enrichment of the media with factors influencing secretion (Figure 1). However, primary cortical neurons were not viable in the presence of Opti-MEM, whereas they survived well in Neurobasal medium, regardless of the addition of BSA-containing B27 supplement (Table 1). Since high amounts of B27 supplement lead to the appearance of BSA accumulates in the region of the membrane where mHtt normally migrates (even when BSA was partially removed by immunoprecipitation) (Figure 2), we opted for Neurobasal media with reduced amount of B27 (0.2%).

To address the issue of low abundance of mHtt in the media, we compared several approaches to concentrate the media and thus enrich mHtt. While TCA precipitation of proteins and immunoprecipitation of mHtt from the media did not give satisfactory results, ultrafiltration of the media proved to be the method of choice for both Neuro2A cells and neurons. We were then able to detect mHtt by immunoblotting (Figure 1).


Figure 1. Optimizing conditions for detection of mHtt secreted from Neuro2A-mHtt cells. 800,000 mHtt-Neuro2A cells were incubated for 4 h in the following media: DMEM, Opti-MEM, conditioned Opti-MEM and HBSS. Cells were lysed, and media were collected, concentrated and analyzed by SDS-PAGE/immunoblotting using anti-Htt antibody. Tubulin was used as a loading control for the cell lysates. The fifth lane represents concentrated conditioned media before being applied on mHtt-expressing Neuro2A.

Table 1. Cells from Figure 2 were visually monitored for viability



Figure 2. Optimizing conditions for detection of mHtt in primary cortical neurons. 700,000 cells were incubated overnight in Neurobasal media with 2% B27 supplement (NB 2% B27), plain Neurobasal media (NB), Opti-MEM, Neurobasal media with 0.4 or 0.2% B27, a mix of Opti-MEM and Neurobasal media at 1:1, or Neurobasal media with 2% B27 where BSA was partially depleted by immunoprecipitation using anti-BSA agarose beads (NB 2% B27 -BSA). The media were collected, concentrated and analyzed by SDS-PAGE/Western blotting. Membrane was stained by Ponceau S to reveal proteins. Large accumulations of proteins (BSA) in the region where mHtt normally migrates (arrow) revealed that the conditions for lanes 1 and 7 were not optimal for mHtt-immunodetection.

Materials and Reagents

  1. Pipette tips (Genesee Scientific, Olympus Plastics, catalog numbers: 24-412 , 24-430 , 24-401 )
  2. 100 mm dishes (Corning, catalog number: 430167 )
  3. 60 mm dishes (Corning, catalog number: 430166 )
  4. 12-well plates (Corning, catalog number: 3513 )
  5. 6-well plates (Corning, catalog number: 3516 )
  6. 1.5 ml centrifugation tubes (Fisher Scientific, Fisherbrand, catalog number: 05-408-129 )
  7. Amicon Ultra-0.5 centrifugal filter units, NMWL 10 (Millipore Sigma, catalog number: UFC501096 )
  8. 15 ml conical tubes (DOT Scientific, catalog number: 229411 )
  9. 50 ml conical tubes (DOT Scientific, catalog number: 229421 )
  10. Cell strainer (70 µm, Corning, Falcon®, catalog number: 352350 )
  11. NuPAGE® Novex® 3-8% Tris-Acetate Gels, 1.0 mm, 15-well (Thermo Fisher Scientific, InvitrogenTM, catalog number: EA03755BOX )
  12. Neuro2A cells (ATCC, catalog number: CCL-131 )
  13. Neuro2A-mHtt cells (stably expressing fragment of mHtt from amino acid 1-571 containing 72 glutamines) (Trajkovic et al., 2017)
  14. mHtt-Flag lentivirus (lentivirus encoding for a fragment of mHtt from amino acid 1-571 containing 72 glutamines with a C-terminal Flag tag)
  15. Sprague Dawley embryos E18, both sexes (Charles River Laboratories)
  16. Poly-D-lysine (Sigma-Aldrich, catalog number: P1149 )
  17. Boric acid (Sigma-Aldrich, catalog number: B7660 )
  18. Borax (Sigma-Aldrich, catalog number: B9876 )
  19. Sodium hydroxide (NaOH) (Avantor Performance Materials, catalog number: 5000-02 )
  20. DMEM (4.5 g/L Glucose, L-Glutamine, Sodium Pyruvate; Thermo Fisher Scientific, InvitrogenTM, catalog number: 11995073 )
  21. Opti-MEM (Thermo Fisher Scientific, InvitrogenTM, catalog number: 31985088 )
  22. Fetal bovine serum, certified, US origin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16000044 )
  23. 4x Laemmli sample buffer (Bio-Rad Laboratories, catalog number: 1610747 )
  24. 2x Laemmli sample buffer (Bio-Rad Laboratories, catalog number: 1610737XTU )
  25. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  26. Milk (Nestle Carnation instant nonfat dry milk)
  27. TBST (20x, with 2% Tween-20, pH 7.4) (Boston Bio Products, catalog number: IBB-180X )
  28. Anti-huntingtin antibody MAB5490 (Millipore Sigma, catalog number: MAB5490 )
  29. Peroxidase-AffiniPure Goat Anti-mouse IgG (H+L) (min X Hu, Bov, Hrs, Rb, Sw Sr Prot) (Jackson ImmunoResearch, catalog number: 115-035-146 )
  30. SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, catalog number: 34096 )
  31. 10x HBSS (Thermo Fisher Scientific, InvitrogenTM, catalog number: 14185052 )
  32. HEPES (Sigma-Aldrich, catalog number: H4034 )
  33. 100 mM sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  34. D-glucose (Sigma-Aldrich, catalog number: G7021 )
  35. Penicillin-streptomycin, 10,000 U/ml (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15140122 )
  36. Trypsin 2.5% (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15090046 )
  37. Neurobasal media (Thermo Fisher Scientific, InvitrogenTM, catalog number: 21103049 )
  38. B27 supplement (50x) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 17504044 )
  39. L-glutamine, 200 mM (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25030081 )
  40. Ponceau S solution (Sigma-Aldrich, catalog number: P7170-1L )
  41. HIV-1 p24 Antigen ELISA (ZeptoMetrix, catalog number: 0 801111 )
  42. PierceTM LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, catalog number: 88953 )
  43. 0.1 M borate buffer (see Recipes)
  44. DMEM/10% heat-inactivated FBS (see Recipes)
  45. 0.3 M HEPES (see Recipes)
  46. HBSS (see Recipes)
  47. Serum media (see Recipes)
  48. Full neuronal media (see Recipes)
  49. Neuronal secretion media (see Recipes)

Equipment

  1. Automatic pipettes (Gilson, model: Pipetman® L, catalog number: F167370 )
  2. FormaTM Steri-CycleTM CO2 Incubator, 37 °C (Thermo Fisher Scientific, model: FormaTM Steri-CycleTM )
  3. S-500 orbital shaker (VWR, model: S-500 , catalog number: 14005-830)
  4. Eppendorf® refrigerated centrifuge (Eppendorf, model: 5417 R )
  5. Allegra X-30 centrifuge (Beckman Coulter, model: Allegra® X-30 )
  6. Bio-Rad Trans-Blot® TurboTM Transfer System (Bio-Rad Laboratories, model: Trans-Blot® TurboTM Transfer System )
  7. ChemiDocTM XRS+ System (Bio-Rad Laboratories)

Software

  1. ImageJ software
  2. Image LabTM Software for ChemiDocTM XRS+ System (Bio-Rad Laboratories)

Procedure

  1. Detection by immunoblotting of mutant huntingtin secreted from Neuro2A cells (Figure 3)


    Figure 3. Schematic representation of the protein secretion assay

    1. Coat each well of a 12-well plate with 1 ml poly-D-lysine (50 µg/ml in 0.1 M borate buffer [see Recipes]) for one hour at room temperature (RT); wash the wells two times with 1 ml autoclaved Milli Q water (RT).
    2. Plate 800,000 of Neuro2A-mHtt cells onto each well and add DMEM/10% heat-inactivated FBS (37 °C; see Recipes) to a total volume of 1 ml.
    3. Place the cells in an incubator with 5% CO2 at 37 °C and incubate them for 5-6 h.
    4. During this time, prepare conditioned media using a 100 mm dish containing a confluent layer of naïve Neuro2A cells (Neuro2A not expressing mHtt) grown in 10 ml DMEM/10% heat-inactivated FBS. Replace the media with 10 ml of Opti-MEM and incubate the cells for 4 h in the incubator with 5% CO2 at 37 °C. Collect the resulting conditioned Opti-MEM and use immediately in Step A5.
    5. Replace the media from Neuro2A-mHtt cells from Step A3 with 800 μl of freshly conditioned Opti-MEM from Step A4 and incubate the cells for additional 1-4 h. Before analysis of mHtt secretion, transfer the media from each well to individual 1.5 ml centrifugation tubes and place the tubes on ice. Lyse the cells on ice in 200 µl of 2x Laemmli buffer with 5% 2-mercaptoethanol, transfer the lysates to 1.5 ml centrifugation tubes and store them on ice while processing the media.
    6. Centrifuge the media at 2,000 x g for 20 min to remove cell debris. Transfer 500 µl of the cleared supernatant to a centrifugal filter unit and centrifuge for 30 min at 14,000 x g. Transfer the resulting concentrated media (~40 μl) to a clean centrifugation tube and add ⅓ of its volume (13.3 μl) of 4x Laemmli buffer with 10% 2-mercaptoethanol. Handle the media and perform all centrifugation steps at 4 °C.
    7. Boil cell lysates and the supernatants for 5 min and then place them on ice.
    8. Load 15 μl per well of the concentrated media in Laemmli buffer and 8 μl per well of cell lysates on 3-8% Tris-Acetate gels. Separate proteins by SDS-PAGE. Transfer the proteins from the gels onto nitrocellulose membranes using Bio-Rad Trans-Blot® TurboTM Transfer System.
    9. Incubate the membranes for 1 h in 5% milk dissolved in TBS-Tween (RT), then overnight in anti-Htt antibody dissolved at 1:1,000 in 5% BSA/TBS-Tween (4 °C). Wash the membrane 3 x 5 min in TBS-Tween and incubate for further 2 h in the secondary antibody diluted at 1:3,000 in TBS-Tween (RT). Wash the membrane 3 x 5 min in TBS-Tween (RT). Perform all washes and incubations with milk and antibodies on a shaker, level 2. Expose the membrane to West Femto Substrate and reveal mHtt using ChemiDoc imaging system.
    10. A representative example of the data obtained using this protocol is illustrated in Figure 1.

  2. Detection by Western blotting of mutant huntingtin secreted from primary cortical neurons
    Preparation of primary cortical neurons
    1. Coat each well of 6-well plates with 2 ml of poly-D-lysine (50 µg/ml in 0.05 M borate buffer [see Recipes]) overnight (RT). Wash each well with 2 ml autoclaved Milli Q water 1 x 5 min, 1 x 1 h, 1 x overnight, then 2 x 1 h on the next day (RT). Do not let the plates dry at any time. Fill each well with 1 ml serum media (see Recipes).
    2. Extract brains from rat embryos at E18 and dissect them in HBSS (see Recipes). Remove meninges and place cortices in 5 ml of HBSS in 60 mm dishes on ice.
    3. Chop the tissue into small pieces in HBSS in the tissue culture hood. Transfer the chopped tissue together with HBSS to a 15 ml conical tube. This and the following steps should be performed at RT, unless indicated otherwise.
    4. Let the tissue fall to the bottom, aspirate most of the supernatant, add 10 ml HBSS and repeat this step 3-4 times. Last time, leave 4.5 ml of the supernatant, add 0.5 ml 2.5% trypsin and mix by flipping the tube. Incubate for 15 min at 37 °C.
    5. Add 5 ml HBSS and gently mix by flipping the tube. Remove all but 2 ml of the supernatant using a pipette.
    6. Dissociate the tissue by pipetting up and down 10 x using P1000 pipette. Filter the obtained cell suspension using a cell strainer and collect the filtrate into a 50 ml conical tube. Rinse the cell strainer with 4 ml HBSS and collect the filtrate into the same tube.
    7. Transfer the filtrate into a 15 ml conical tube and spin at 500 x g for 5 min at RT. Aspirate the supernatant.
    8. The pellet will be mostly white, with some red cells at the bottom. To ensure the purity of the culture, dislodge only the white part of the pellet by tapping the tube (red cells are more compact and will remain attached to the bottom of the tube) and resuspend the cells in 4 ml/brain of serum media within the same tube (see Recipes). Transfer the cell suspension into a new conical tube.
    9. Plate 700,000 cells per well in poly-D-lysine-coated 6-well plates with a total of 2 ml serum media.
    10. After 2 h, replace the serum media with 2 ml of full neuronal media (see Recipes).

    Detection of mutant huntingtin by Western blotting
    1. At DIV (days in vitro) 3, transduce primary cortical neurons with mHtt-Flag lentivirus at 5 MOI (virus equivalent of 700 ng p24; 1 ng p24 contains 5,000 infectious particles).
    2. After 24 h, replace the media with full neuronal media.
    3. At DIV 10, replace the media with 1 ml of neuronal secretion media (see Recipes).
    4. Collect the media after overnight incubation; concentrate and analyze the media as in Procedure A.
    5. Lyse the cells directly in 500 µl of 2x Laemmli buffer.
    6. For representative examples of the results obtained using this protocol, please see Trajkovic et al., 2017.

Data analysis

The intensity of bands was determined using ‘Analyze gels’ module of ImageJ software. The ratio between the extracellular and intracellular protein was determined. All experiments were repeated at least three times. Significance was determined using unpaired t-test.

Notes

Since proteins present in the cell culture media may be bona fide secreted proteins as well as proteins released due to spontaneous cell lysis and toxic effects of some reagents used to treat the cells, it is advisable to perform an LDH cytotoxicity assay on the media collected from control and treated cells. To that end use LDH Cytotoxicity Assay Kit according to manufacturer’s instruction. Only treatments that do not cause significant toxicity should be considered for further analysis.

Recipes

Note: All solutions should be stored at 4 °C.

  1. 0.05 M borate buffer
    1.24 g boric acid
    1.90 g borax
    Dissolved in total 500 ml Milli Q H2O
    Adjust pH to 8.5 using 1 N NaOH; filter sterilize
  2. DMEM/10% HI FBS
    Add 50 ml of heat-inactivated FBS to 450 ml DMEM
  3. 0.3 M HEPES
    7.15 g HEPES
    ~90 ml H2O
    Adjust pH to 7.3 using 10 N and 1 N NaOH
    Add Milli Q H2O to 100 ml final volume
  4. HBSS
    50 ml 10x HBSS
    16.5 ml 0.3 M HEPES (see above)
    5 ml 100 mM sodium pyruvate
    3 g D-glucose (Sigma-Aldrich)
    5 ml penicillin/streptomycin stock
    Add Milli Q water to the final volume 500 ml; filter sterilize
  5. Serum media
    44 ml Neurobasal media
    5 ml HI FBS
    0.5 ml penicillin/streptomycin stock
    0.5 ml glutamine stock
  6. Full neuronal media
    10 ml B27 supplement
    5 ml penicillin/streptomycin stock
    5 ml glutamine stock
    Added to 480 ml Neurobasal medium
  7. Neuronal secretion media
    1 ml B27 supplement
    5 ml penicillin/streptomycin stock
    5 ml glutamine stock
    Added to 489 ml Neurobasal medium

Acknowledgments

This work has been supported by R01NS080331 (D.K.). This protocol has been originally published in Journal of Neuroscience (Trajkovic et al., 2017). The authors declare no conflict of interest.

References

  1. Chevallet, M., Diemer, H., Van Dorssealer, A., Villiers, C. and Rabilloud, T. (2007). Toward a better analysis of secreted proteins: the example of the myeloid cells secretome. Proteomics 7(11): 1757-1770.
  2. Lee, M. C., Miller, E. A., Goldberg, J., Orci, L. and Schekman, R. (2004). Bi-directional protein transport between the ER and Golgi. Annu Rev Cell Dev Biol 20: 87-123.
  3. Momen-Heravi, F., Balaj, L., Alian, S., Mantel, P. Y., Halleck, A. E., Trachtenberg, A. J., Soria, C. E., Oquin, S., Bonebreak, C. M., Saracoglu, E., Skog, J. and Kuo, W. P. (2013). Current methods for the isolation of extracellular vesicles. Biol Chem 394(10): 1253-1262.
  4. Trajkovic, K., Jeong, H. and Krainc, D. (2017). Mutant huntingtin is secreted via a late endosomal/lysosomal unconventional secretory pathway. J Neurosci 37(37): 9000-9012.
  5. Zhang, M. and Schekman, R. (2013). Cell biology. Unconventional secretion, unconventional solutions. Science 340(6132): 559-561.

简介

由细胞分泌的蛋白质的定量分析由于它们的丰度低和在细胞培养基中干扰大量牛血清白蛋白(BSA)的存在而提出挑战。 我们建立检测突变亨廷顿蛋白(mHtt)检测分泌Neuro2A细胞系稳定表达mHtt和大鼠原代皮层神经元蛋白质印迹。 我们的方案是基于降低培养基中BSA的量,同时保持细胞活力和分泌潜能,并在通过超滤分析之前浓缩培养基。


【背景】许多蛋白质通过各种分泌途径从细胞分泌到细胞外环境中。这些途径包括在ER-高尔基体 - 质膜途径之后的常规分泌途径(Lee等,2004)和多个非常规途径,例如溶酶体胞吐作用,穿过质膜的易位和外泌体以及胞外体释放(Zhang和Schekman,2013)。为了研究这些途径,经常需要分析培养细胞分泌的蛋白质进入培养基。蛋白质可以游离形式分泌,也可以与胞膜结构如胞外体和外泌体结合(Zhang and Schekman,2013)。这些蛋白质是否与膜相关联,决定了分析方法的类型。为了分离膜相关蛋白质,通常使培养基经受差速离心程序,在其上沉积含蛋白质的膜,浓缩并从培养基的成分中纯化,这允许不受限制的蛋白质分析(Momen-Heravi等人。,2013)。然而,自由形式蛋白质的分析是一个更具挑战性的任务(Chevallet等人,2007)。两个主要障碍是1)在大多数细胞培养基中加入大量的牛血清白蛋白(BSA),这可能掩盖感兴趣的蛋白质;和2)至少一些分泌蛋白包括mHtt在介质中低丰度。为了研究分泌的mHtt,开发了该方案,并且可以用于分析培养基中的其他低丰度,自由形式的蛋白质。

协议优化
为了解决过度BSA的问题,我们首先监测不同培养基中的细胞活力,部分或全部减少BSA。在Neuro2A细胞稳定表达72个谷氨酸(Neuro2A-mHtt细胞)的mHtt的N-末端571个氨基酸的情况下,最佳培养基为Opti-MEM,其不仅保存细胞活力,而且增强突变体的分泌亨廷顿蛋白(mHtt)与其他培养基比如没有补充胎牛血清(FBS)和HBSS(图1)的DMEM相比较。此外,我们观察到应用条件Opti-MEM(与不表达mHtt的幼稚Neuro2A细胞预孵育的Opti-MEM)显着增强了来自Neuro2A-mHtt细胞的mHtt的分泌,可能是由于培养基富集影响分泌的因子(图1)。然而,在Opti-MEM存在的情况下,原代皮质神经元不能存活,而在Neurobasal培养基中存活良好,无论添加含BSA的B27补充物如何(表1)。由于大量的B27补充剂导致BSA的出现在mHtt正常迁移的膜区域积累(即使当通过免疫沉淀部分去除BSA时)(图2),我们选择B27减少的Neurobasal培养基(0.2 %)。

为了解决媒体中媒体资源匮乏的问题,我们比较了几种方法来集中媒体,从而丰富媒体。尽管TCA沉淀蛋白质并且从培养基中免疫沉淀mHtt并没有给出令人满意的结果,但是对于Neuro2A细胞和神经元两者而言,介质的超滤被证明是选择的方法。然后我们能够通过免疫印迹检测mHtt(图1)。

“”src
图1.优化用于检测从Neuro2A-mHtt细胞中分泌的mHtt的条件将800,000个mHtt-Neuro2A细胞在以下培养基中培养4小时:DMEM,Opti-MEM,条件化的Opti-MEM和HBSS 。裂解细胞,收集培养基,浓缩并通过使用抗Htt抗体的SDS-PAGE /免疫印迹进行分析。使用微管蛋白作为细胞裂解物的上样对照。第五泳道代表浓缩的条件培养基,然后应用于表达mHtt的Neuro2A。

表1.图2中的细胞用肉眼观察生存力


“”src
图2.优化用于检测原代皮层神经元中mHtt的条件将700,000个细胞在含有2%B27添加物(NB 2%B27)的Neurobasal培养基,平原Neurobasal培养基(NB),Opti- MEM,具有0.4或0.2%B27的Neurobasal培养基,1:1的Opti-MEM和Neurobasal培养基的混合物,或2%B27的Neurobasal培养基,其中使用抗BSA琼脂糖珠(NB 2%B27 -BSA)。收集培养基,浓缩并通过SDS-PAGE / Western印迹分析。膜被Ponceau S染色以显示蛋白质。在mHtt正常迁移的区域(箭头),大量蛋白质(BSA)显示泳道1和7的条件不是最佳的

关键字:蛋白质分泌, 超滤, BSA

材料和试剂

  1. 移液器吸头(Genesee Scientific,Olympus Plastics,目录号:24-412,24-430,24-401)
  2. 100毫米的菜肴(康宁,目录号:430167)
  3. 60毫米盘子(康宁,目录号:430166)
  4. 12孔板(康宁,目录号:3513)
  5. 6孔板(康宁,目录号:3516)
  6. 1.5ml离心管(Fisher Scientific,Fisherbrand,目录编号:05-408-129)
  7. Amicon Ultra-0.5离心过滤装置,NMWL 10(Millipore Sigma,目录号:UFC501096)
  8. 15 ml锥形管(DOT Scientific,目录号:229411)
  9. 50毫升锥形管(DOT Scientific,目录号:229421)
  10. 细胞过滤器(70μm,Corning,Falcon ,目录号:352350)
  11. NuPAGENovex3-8%Tris-乙酸盐凝胶,1.0mm,15孔(Thermo Fisher Scientific,Invitrogen TM,产品目录号:EA03755BOX)
  12. Neuro2A细胞(ATCC,目录号:CCL-131)
  13. Neuro2A-mHtt细胞(稳定表达来自含有72个谷氨酰胺的氨基酸1-571的mHtt片段)(Trajkovic等人,2017)
  14. mHtt-Flag慢病毒(编码来自氨基酸1-571的mHtt片段的慢病毒,含有72个具有C端Flag标签的谷氨酰胺)
  15. Sprague Dawley胚胎E18,两性(Charles River Laboratories)
  16. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P1149)
  17. 硼酸(Sigma-Aldrich,目录号:B7660)
  18. 硼砂(Sigma-Aldrich,目录号:B9876)
  19. 氢氧化钠(NaOH)(Avantor Performance Materials,目录号:5000-02)
  20. DMEM(4.5g / L葡萄糖,L-谷氨酰胺,丙酮酸钠; Thermo Fisher Scientific,Invitrogen TM,目录号:11995073)
  21. Opti-MEM(Thermo Fisher Scientific,Invitrogen TM,目录号:31985088)
  22. 来源于美国的胎牛血清(Thermo Fisher Scientific,Invitrogen TM,产品目录号:16000044)
  23. 4×Laemmli样品缓冲液(Bio-Rad Laboratories,目录号:1610747)
  24. 2x Laemmli样品缓冲液(Bio-Rad Laboratories,目录号:1610737XTU)
  25. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  26. 牛奶(雀巢康乃馨即时脱脂奶粉)
  27. TBST(20x,含2%Tween-20,pH7.4)(Boston Bio Products,目录号:IBB-180X)
  28. 抗亨廷顿蛋白抗体MAB5490(Millipore Sigma,目录号:MAB5490)
  29. (H + L)(min X Hu,Bov,Hrs,Rb,Sw Sr Prot)(Jackson ImmunoResearch,目录号:115-035-146)
    过氧化物酶-AfeiniPure山羊抗小鼠IgG
  30. SuperSignal TM西Femto最大灵敏度底物(Thermo Fisher Scientific,目录号:34096)
  31. 10×HBSS(Thermo Fisher Scientific,Invitrogen TM,目录号:14185052)
  32. HEPES(Sigma-Aldrich,目录号:H4034)
  33. 100mM丙酮酸钠(Thermo Fisher Scientific,Gibco TM,产品目录号:11360070)
  34. D-葡萄糖(Sigma-Aldrich,目录号:G7021)
  35. 10,000U / ml青霉素 - 链霉素(Thermo Fisher Scientific,Invitrogen TM,目录号:15140122)
  36. 胰蛋白酶2.5%(Thermo Fisher Scientific,Invitrogen TM,目录号:15090046)
  37. 神经基础培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:21103049)
  38. B27添加物(50x)(Thermo Fisher Scientific,Invitrogen TM,目录号:17504044)
  39. L-谷氨酰胺,200mM(Thermo Fisher Scientific,Invitrogen TM,目录号:25030081)
  40. Ponceau S溶液(Sigma-Aldrich,目录号:P7170-1L)
  41. HIV-1 p24抗原ELISA(ZeptoMetrix,目录号:0801111)
  42. Pierce TM LDH细胞毒性测定试剂盒(Thermo Fisher Scientific,目录号:88953)
  43. 0.1 M硼酸缓冲液(见食谱)
  44. DMEM / 10%热灭活FBS(见食谱)
  45. 0.3 M HEPES(见食谱)
  46. HBSS(见食谱)
  47. 血清培养基(见食谱)
  48. 完整的神经元媒体(见食谱)
  49. 神经元分泌介质(见食谱)

设备

  1. 自动移液器(Gilson,型号:Pipetman®L,产品目录号:F167370)
  2. Forma TM TM Steri-Cycle TM CO 2培养箱,37℃(Thermo Fisher Scientific,型号:Forma TM TM) Steri-Cycle )
  3. S-500轨道摇床(VWR,型号:S-500,目录号:14005-830)
  4. Eppendorf冷冻离心机(Eppendorf,型号:5417 R)
  5. Allegra X-30离心机(Beckman Coulter,型号:Allegra X-30)
  6. Bio-Rad Trans-Blot TM Turbo TM转移系统(Bio-Rad Laboratories,型号:Trans-Blot TM Turbo TM TM) / sup>传输系统)
  7. ChemiDoc TM XRS +系统(Bio-Rad Laboratories)

软件

  1. ImageJ软件
  2. 用于ChemiDoc TM XRS +系统(Bio-Rad Laboratories)的Image Lab TM软件

程序

  1. 通过免疫印迹从Neuro2A细胞分泌的突变体亨廷顿蛋白的检测(图3)


    图3.蛋白质分泌测定的示意图

    1. 在室温(RT)下,用1ml聚-D-赖氨酸(在0.1M硼酸盐缓冲液中50μg/ ml [参见配方])将每孔的12孔板包衣1小时;用1ml高压灭菌的Milli Q水(RT)洗两次孔。
    2. 将800,000个Neuro2A-mHtt细胞铺在每个孔上并加入DMEM / 10%热灭活的FBS(37℃;参见食谱)至总体积1ml。
    3. 将细胞置于含有5%CO 2的培养箱中37°C孵育5-6小时。
    4. 在此期间,使用含有在10ml DMEM / 10%热灭活的FBS中生长的幼稚Neuro2A细胞(Neuro2A不表达mHtt)的100mm培养皿制备条件培养基。用10ml的Opti-MEM取代培养基,并在37℃下在具有5%CO 2的培养箱中孵育细胞4小时。收集得到的条件Opti-MEM并立即在步骤A5使用。
    5. 用来自步骤A4的800μl新鲜调理的Opti-MEM替换来自步骤A3的Neuro2A-mHtt细胞的培养基,并再孵育细胞1-4小时。在分析mHtt分泌物之前,将各孔的培养基转移至各1.5ml离心管中并置于冰上。在200μl含有5%2-巯基乙醇的2x Laemmli缓冲液中将细胞溶解在冰上,将裂解物转移到1.5ml离心管中,并在处理培养基时将其储存在冰上。
    6. 将培养基以2000×g离心20分钟以除去细胞碎片。将500μl澄清的上清液转移至离心过滤装置,并以14,000×gg离心30分钟。将所得到的浓缩培养基(〜40μl)转移至干净的离心管中,加入其体积为13.3μl的4倍Laemmli缓冲液(含10%2-巯基乙醇)。处理培养基并在4°C下进行所有离心步骤。
    7. 煮沸细胞裂解物和上清液5分钟,然后放置在冰上。
    8. 在Laemmli缓冲液中加入15μl每孔的浓缩培养基,在3-8%Tris-乙酸盐凝胶上每孔加入8μl细胞裂解物。通过SDS-PAGE分离蛋白质。使用Bio-Rad Trans-Blot TM Turbo Transfer TM系统将凝胶中的蛋白质转移到硝酸纤维素膜上。
    9. 在溶于TBS-Tween(RT)中的5%牛奶中孵育膜1h,然后在5%BSA / TBS-Tween(4℃)中以1:1,000溶解的抗Htt抗体过夜。在TBS-Tween中洗膜3次,每次5分钟,并在TBS-Tween(RT)中以1:3,000稀释的二次抗体中继续孵育2小时。在TBS-Tween(RT)中洗膜3×5分钟。在2级振荡器上用牛奶和抗体进行所有洗涤和孵育。将膜暴露于West Femto Substrate,并使用ChemiDoc成像系统显示mHtt。
    10. 图1说明了使用该协议获得的数据的一个代表性例子。

  2. 蛋白质印迹法检测初级皮层神经元分泌的突变亨廷顿蛋白 初级皮质神经元的制备
    1. 用2ml聚-D-赖氨酸(在0.05M硼酸盐缓冲液中50μg/ ml [参见配方])将每孔的6孔板过夜(RT)包被。用2ml高压灭菌的Milli Q水1×5分钟,1×1小时,1×过夜,然后在第二天(RT)2×1小时洗涤每个孔。不要让盘子随时干燥。用1ml血清培养基填充每个孔(见食谱)。
    2. 在E18处提取大鼠胚胎的大脑并将其解剖在HBSS中(见食谱)。去除脑膜并将皮质放置在冰上60毫米的5毫升HBSS中。
    3. 在组织培养罩中将组织切成HBSS中的小块。将切碎的组织与HBSS一起转移到15ml锥形管中。
      除非另有说明,否则以下步骤应在室温下进行
    4. 让组织下降到底部,吸出大部分上清液,加入10毫升HBSS,重复这一步3-4次。最后一次,离开4.5毫升的上清,加入0.5毫升2.5%胰蛋白酶和翻转管混合。
      在37°C孵育15分钟
    5. 加入5毫升HBSS轻轻混合,翻转管。
      用移液管除去2毫升上清液
    6. 使用P1000移液器上下移液10次以分离组织。使用细胞过滤器过滤获得的细胞悬液并将滤液收集到50ml锥形管中。用4毫升HBSS冲洗细胞过滤器,并将滤液收集到相同的管中。
    7. 将滤液转移到15ml锥形管中,并在室温下500×g旋转5分钟。吸出上清液。
    8. 颗粒大部分是白色的,底部有一些红色的细胞。为了确保培养物的纯度,通过轻敲管子(红细胞更紧凑并且将保持连接到管的底部)仅移出颗粒的白色部分,并将细胞重悬于4ml /血清培养基的脑中相同的管(见食谱)。将细胞悬液转移到新的锥形管中。
    9. 在含有总共2ml血清培养基的聚-D-赖氨酸包被的6孔板中每孔铺板700,000个细胞。
    10. 2小时后,用2ml完全神经元培养基替换血清培养基(见食谱)。

    Western blotting检测突变亨廷顿蛋白
    1. 在DIV(天体外实验)3中,以5MOI(病毒当量为700ng p24; 1ng p24含有5000个感染性颗粒)转导具有mHtt-Flag慢病毒的原代皮层神经元。
    2. 24小时后,用全神经元培养基更换培养基。
    3. 在DIV10,用1ml神经元分泌介质替换培养基(见食谱)。
    4. 过夜培养后收集培养基;集中和分析程序A中的媒体。
    5. 将细胞直接溶解在500μl2x Laemmli缓冲液中。
    6. 对于使用此协议获得的结果的代表性实例,请参阅2017年的Trajkovic 等。。

数据分析

使用ImageJ软件的“分析凝胶”模块确定条带的强度。测定细胞外和细胞内蛋白质的比例。所有实验重复至少三次。意义是使用不成对的 t - 测试确定的。

笔记

由于存在于细胞培养基中的蛋白质可能是真正分泌的蛋白质以及由于自发性细胞溶解和用于治疗细胞的一些试剂的毒性作用释放的蛋白质,建议进行LDH对从对照和处理的细胞收集的培养基进行细胞毒性测定。为此,根据生产商的说明使用LDH细胞毒性分析试剂盒。只有不引起显着毒性的治疗才应被考虑进一步分析。

食谱

注意:所有的解决方案应该保存在4°C。

  1. 0.05 M硼酸盐缓冲液
    1.24克硼酸
    1.90克硼砂
    溶于总共500ml Milli Q H 2 O
    用1N NaOH调节pH至8.5;过滤消毒
  2. DMEM / 10%HI FBS
    添加50毫升热灭活FBS到450毫升DMEM
  3. 0.3 M HEPES
    7.15克HEPES
    〜90ml H 2 O 使用10 N和1 N NaOH将pH调节至7.3 加Milli Q H 2 O至100ml最终体积
  4. HBSS
    50毫升10倍HBSS

    16.5 ml 0.3 M HEPES(见上文) 5毫升100毫克丙酮酸钠
    3克D-葡萄糖(西格玛奥德里奇)
    5毫升青霉素/链霉素库存
    加入Milli Q水至最终体积500 ml;过滤消毒
  5. 血清培养基
    44毫升Neurobasal媒体
    5毫升HI FBS
    0.5毫升青霉素/链霉素库存
    0.5毫升谷氨酰胺股票
  6. 完整的神经元媒体
    10毫升B27补充
    5毫升青霉素/链霉素库存
    5毫升谷氨酰胺库存
    添加到480毫升Neurobasal培养基
  7. 神经元分泌介质
    1毫升B27补充
    5毫升青霉素/链霉素库存
    5毫升谷氨酰胺库存
    添加到489毫升Neurobasal培养基

致谢

这项工作得到了R01NS080331(D.K.)的支持。该协议最初发表于“神经科学杂志”(Journal of Neuroscience)(Trajkovic等人,2017)。作者宣称没有利益冲突。

参考

  1. Chevallet,M.,Diemer,H.,Van Dorssealer,A.,Villiers,C。和Rabilloud,T。(2007)。 更好地分析分泌的蛋白质:髓细胞分泌组的例子 < em> Proteomics 7(11):1757-1770。
  2. Lee,M.C.,Miller,E.A.,Goldberg,J.,Orci,L。和Schekman,R。(2004)。 雌激素与高尔基体之间的双向蛋白转运 Annu Rev Cell Dev Biol 20:87-123。
  3. Momen-Heravi,F.,Balaj,L.,Alian,S.,Mantel,PY,Halleck,AE,Trachtenberg,AJ,Soria,CE,Oquin,S.,Bonebreak,CM,Saracoglu,E.,Skog,J 。和Kuo,WP(2013)。 目前分离细胞外囊泡的方法 Biol Chem > 394(10):1253-1262。
  4. Trajkovic,K.,Jeong,H.和Krainc,D。(2017)。 突变型亨廷顿蛋白是通过晚期内体/溶酶体非常规分泌途径分泌的。 J Neurosci 37(37):9000-9012。
  5. 张,M.和Schekman,河(2013)。 细胞生物学。非传统的分泌,非传统的解决方案。 科学 340(6132):559-561。
  • 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。
Copyright: © 2018 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. Trajkovic, K., Jeong, H. and Krainc, D. (2018). Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons. Bio-protocol 8(1): e2675. DOI: 10.21769/BioProtoc.2675.
  2. Trajkovic, K., Jeong, H. and Krainc, D. (2017). Mutant huntingtin is secreted via a late endosomal/lysosomal unconventional secretory pathway. J Neurosci 37(37): 9000-9012.
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