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Separation of Intracellular Vesicles for Immunoassays
分离胞内囊泡用于免疫分析   

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参见作者原研究论文

本实验方案简略版
Immunity
Sep 2014

Abstract

The endosome/lysosome systems play important roles in immune cell functions as signaling platforms. Immune cells utilize these endosome/lysosome for signal transduction or intercellular communication to elicit the proper immune responses, regulating the localization or the association of the signaling complexes. Here we introduce the procedures to separate the intracellular vesicles such as endosomes or lysosomes, which could be useful to identify the subcellular localization of the signaling complexes.

Materials and Reagents

  1. Cells (e.g. RAW264.7, Raji, mouse naïve B cells)
  2. Trypan blue staining (0.4% solution) (Life Technologies, catalog number: 15250-061 )
  3. CD43-microbeads (Miltenyi Biotec, catalog number: 130-049-801 )
  4. Blocking One (Nacalai tesque, catalog number: 03953-95 )
  5. Anti-mouse LAMP1 (eBioscience, catalog number: 1D4B )
  6. Anti-GM130 (BD biosciences, catalog number: 35/GM130 )
  7. Anti-Rab5 (Santa Cruz Biotechnologies, catalog number: D-11 )
  8. Anti-Calnexin (Enzo Life Sciences, catalog number: ADI-SPA-860 )
  9. Anti-COX IV (Cell Signaling Technology, catalog number: 4844 )
  10. Anti-rat Ig(H+L)-HRP (Jackson ImmunoResearch Laboratories, catalog number: 712-036-153 )
  11. Anti-mouse Ig(H+L)-HRP (Jackson ImmunoResearch Laboratories, catalog number: 115-035-062 )
  12. Anti-rabbit Ig(H+L)-HRP (Jackson ImmunoResearch Laboratories, catalog number: 111-035-144 )
  13. ECL substrate (Thermo Fisher Scientific, catalog number: 34095 )
  14. Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 87785 )
  15. Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 78420 )
  16. 60% OptiPrep (Axis-Shield plc, catalog number: 1114542 )
  17. Solution A (see Recipes)
  18. Solution B (see Recipes)
  19. Solution C (see Recipes)
  20. Solution D (see Recipes)
  21. Solution E (see Recipes)
  22. 30%, 23%, 17%, 11%, and 5% Optiprep (2 ml each) (see Recipes)
  23. TBS-T (see Recipes)

Equipment

  1. 10 cm dish
  2. Centrifuge tube (Beckman Coulter, catalog number: 347357 )
  3. Hemocytometer
  4. Centrifuge (with cooling function will be recommended)
  5. 1 ml syringe with 29-gauge needle

Procedure

Protocol A should be chosen when regular cell-line (e.g. RAW264.7, Raji, or HEK293T) or cells having large cytosol in volume are analyzed whereas Protocol B should be chosen for small cells or cells having small cytosol, such as lymphocytes.

  1. Procedures for cell extraction from 10 cm dish (2 dishes for each sample)
    1. Layer 360 µl of the each gradient solution (30%, 23%, 17%, 11%, and 5% OptiPrep) in the Centrifuge tube(s) with P200 micropipette, as shown in Figure 1.


      Figure 1. Illustration showing the way to layer the gradient solution using P200 micropipette. Pour 360 µl of each gradient solution carefully into a centrifuge tube using P200 micropipette (180 µl x 2 times), not to make bubbles nor disturb the other layers. Layer in a centrifuge tube from the heaviest (30%) to lightest (5%) solution.

    2. Leave tubes at 4 °C for more than one hour and keep it still until use.
    3. Confirm that cells (e.g. RAW264.7, or Raji) are healthy and 75-80% confluent under a microscopy.
    4. (Optional) Treat with stimulants for the desired period (e.g. LPS at 100 ng/ml for 30 min).
    5. Wash twice with 10 ml of ice-cold PBS, transfer to suitable tubes, and determine cell density and viability with a hemacytometer and trypan blue staining.
    6. Transfer 1.5 ~ 2.0 x 107 cells to new tubes and centrifuge at 1,100 rpm for 5 min at 4 °C.
    7. Aspirate the supernatant, tap tubes to loosen cells, and add 450 µl of ice-cold Solution E per tube.
    8. Homogenize cells by 80-100 passages through a 29-gauge needle until cells show roughly 70-80% trypan blue positive. Cells should be kept cold.
    9. Centrifuge the homogenate at 1,000 x g for 5 min at 4 °C and transfer the whole supernatant (the post-nuclear fraction) to new 1.5 ml tubes and centrifuge at 1,000 x g for 5 min at 4 °C again. Repeat this step until pellet of cell debris can not be seen after centrifugation (usually 3 times).
      Note: The whole supernatant should be taken for the second centrifugation, and the same for the third centrifugation. Initial volume of supernatant after centrifugation should be roughly 400 µl, and after 2 times centrifugation it will be reduced to ca. 350 µl.
    10. Carefully layer 300 µl of the supernatant on the top of the gradient solution made in step A1 (Figure 3).
    11. Set the centrifuge tubes in the chilled ultracentrifuge rotor TLS-55.
    12. Centrifuge for 4 h at 39,000 rpm (130,000 x g) at 4 °C by Optima MAX-TL.
      Note: Set acceleration (Accel) and deceleration (Decel) at 8 and 0 (free deceleration), respectively.
    13. Harvest 185 µl per one fraction to make 11 fractions using P200 micropipette, and transfer to 1.5 ml microtubes.
    14. Store at -80 °C until use.

  2. Procedures for mouse naïve B cells
    1. Layer 360 µl of the each gradient solution (30%, 23%, 17%, 11%, and 5% OptiPrep) in the Centrifuge tube(s) with P200 micropipette.
    2. Leave tubes at 4 °C for more than one hour and keep it still until use.
      Separate the naïve splenic B cells by using CD43-microbeads and MACS according to the manufacturer’s instruction (please see the following link: http://www.miltenyibiotec.co.jp/~/media/Images/Products/Import/0001200/IM0001263.ashx).
    3. (Optional) Treat 1.5 ~ 2.0 x 108 of B cells with stimulants for the desired period (e.g. CpG-1668 at 100 nM for 1 h).
    4. Wash twice with 10 ml of ice-cold PBS in suitable tubes, and determine cell density and viability with a hemocytometer and trypan blue staining.
    5. Transfer the cells to new tubes and centrifuge at 1,100 rpm for 5 min at 4 °C.
    6. Aspirate the supernatant, tap tubes to loosen cells, and add 450 µl of ice-cold Solution E per tube.
    7. Homogenize cells by passing roughly 200 times through a 29-gauge needle until cells show 70-80% trypan blue positive. Cells should be kept cold on ice.
    8. Centrifuge the homogenate at 1,000 x g for 5 min at 4 °C and transfer the supernatant (the post-nuclear fraction) to new 1.5 ml tubes. Repeat this step three times.
    9. Carefully layer 300 µl of the supernatant on the top of the gradient.
    10. Set the tubes in the chilled ultracentrifuge rotor TLS-55.
    11. Centrifuge for 4 h at 39,000 rpm (130,000 x g) at 4 °C by Optima MAX-TL.
      Note: Set acceleration (Accel) and deceleration (Decel) at 8 and 0 (free deceleration), respectively.
    12. Harvest 290 µl per one fraction to make 7 fractions using P200 micropipette, and transfer to 1.5 ml microtubes.
    13. Store at -80 °C until use.

  3. Evaluation of the fractionation
    1. Take 30 µl of the fractions and mix with the equal volume of 2x SDS-PAGE sample buffer.
    2. After brief denaturing at 95 °C, apply 15 µl of the samples to run SDS-PAGE and transfer to PVDF membrane.
    3. Block the blot with Blocking One, followed by incubation with the primary antibodies against organelle markers. Following antibodies used in this protocol; anti-LAMP1 (dilution 1:1,000) for the detection of the late endosome/lysosome, anti-GM130 for cis-Golgi, anti-Rab5 for endosome, anti-Calnexin for ER, and anti-COX IV for mitochondria, respectively.
    4. Wash the blots 3 times with TBS-T to remove excess antibodies (5 min x 3 times).
    5. Incubate with the proper secondary antibodies conjugated with HRP for 1 h [Anti-rat Ig (H+L) for anti-LAMP1, anti-mouse Ig (H+L) for anti-GM130 or anti-Rab5, anti-rabbit Ig (H+L) for anti-Calnexin or COX IV, respectively. Concentration of secondary antibodies is roughly at 50 ng/ml].
    6. Wash the blots with TBS-T to remove excess antibodies (15 min x 3 times).
    7. Incubate with ECL substrate, and detect the luminescence signals (Figure 2).


      Figure 2. Intracellular vesicles of mouse naïve B cells were fractionated and their distribution was analyzed by western blotting with the specific antibodies against the following organelle markers; late endosome/lysosome with LAMP1, cis-Golgi with GM130, pan-endosome with Rab5, ER with Calnexin, and mitochondria with COX IV, respectively.

Notes

  1. For homogenization of cells, Dounce homogenizer or other devices can be used, as the degree of homogenization difficulty depends on the cell types.
  2. For small cells such as mouse naïve B cells, number of passage with 29G syringe may differ depending on the cell density.

Recipes

  1. Solution A
    60% OptiPrep
  2. Solution B
    OptiPrep diluent
    (Components)
    100 ml
    (Final conc.)
    H2O
    ~ 42.8 ml

    1 M Hepes (pH7.5)
    15 ml
    150 mM
    1 M KCl
    23.5 ml
    235 mM
    1 M MgCl2
    1.2 ml
    12 mM
    1 M CaCl2
    2.5 ml
    25 mM
    0.2 M EGTA
    15 ml
    30 mM
    Adjust to pH 7.0 with 1 M KOH and make up to 100 ml
  3. Solution C
    40% OptiPrep working solution
    2 vol. of Solution A + 1 vol. of Solution B
  4. Solution D
    Working solution diluent
    (Components) 100 ml (Final conc.)
    (Components)
    100 ml
    (Final conc.)
    H2O
    ~ 81 ml

    1 M Hepes (pH7.5)
    5 ml
    50 mM
    1 M KCl
    7.8 ml
    78 mM
    1 M MgCl2
    400 µl
    4 mM
    1 M CaCl2
    840 µl
    8.4 mM
    0.2 M EGTA
    5 ml
    10 mM
    Adjust to pH 7.0 with 1 M KOH and make up to 100 ml
  5. Solution E
    Homogenization medium
    (Components)
    100 ml
    (Final conc.)
    H2O
    ~ 81 ml

    1 M Hepes (pH7.5)
    5 ml
    50 mM
    1 M KCl
    7.8 ml
    78 mM
    1 M MgCl2
    400 µl
    4 mM
    1 M CaCl2
    840 µl
    8.4 mM
    0.2 M EGTA
    5 ml
    10 mM
    Sucrose (FW 342.30)
    8.56 g
    250 mM
    Adjust to pH 7.0 with 1 M KOH and make up to 100 ml
    Add Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail before use
  6. 30%, 23%, 17%, 11%, and 5% Optiprep (2 ml each)
    (Components)
    30%
    23%
    17%
    11%
    5%
    Solution C (40% Optiprep)
    1500 µl
    1150 µl
    850 µl
    550 µl
    250 µl
    Solution D
    500 µl
    850 µl
    1150 µl
    1450 µl
    1750 µl
    Halt Protease Inhibitor*
    20 µl
    20 µl
    20 µl
    20 µl
    20 µl
    Halt Phosphatase Inhibitor†
    20 µl
    20 µl
    20 µl
    20 µl
    20 µl
    * Halt Protease Inhibitor Cocktail
    † Halt Phosphatase Inhibitor Cocktail


Figure 3. Diagram of completed Opti-prep gradient before ultracentrifugation

  1. TBS-T
    (Components)
    1 L
    (Final conc.)
    1 M Tris-HCl (pH7.5)
    20 ml
    20 mM
    NaCl
    8 g
    137 mM
    Tween 20
    1 ml
    0.1%

Acknowledgments

We thank K. Furuyama-Tanaka and S. Shimabukuro-Demoto for the technical assistance. This protocol was adapted from Axis-shield’s density gradient protocol S44. This work was supported by the Funding Program for Next Generation World-Leading Researchers (Next Program; for N.T.-S., LS134), grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (for N.T.-S., 21390123, for T.K., 25871165), and a grant from the National Center for Global Health and Medicine (for N.T.-S., 23S001).

References

  1. Kobayashi, T., Shimabukuro-Demoto, S., Yoshida-Sugitani, R., Furuyama-Tanaka, K., Karyu, H., Sugiura, Y., Shimizu, Y., Hosaka, T., Goto, M., Kato, N., Okamura, T., Suematsu, M., Yokoyama, S. and Toyama-Sorimachi, N. (2014). The histidine transporter SLC15A4 coordinates mTOR-dependent inflammatory responses and pathogenic antibody production. Immunity 41(3): 375-388.

简介

内体/溶酶体系统在作为信号平台的免疫细胞功能中起重要作用。 免疫细胞利用这些内体/溶酶体进行信号转导或细胞间通讯,以引发适当的免疫应答,调节信号复合物的定位或缔合。 在这里我们介绍分离细胞内囊泡如内体或溶酶体的程序,这可能有助于识别信号复合物的亚细胞定位。

材料和试剂

  1. 细胞(例如 RAW264.7,Raji,小鼠天然B细胞)
  2. 台盼蓝染色(0.4%溶液)(Life Technologies,目录号:15250-061)
  3. CD43微珠(Miltenyi Biotec,目录号:130-049-801)
  4. Blocking One(Nacalai tesque,目录号:03953-95)
  5. 抗小鼠LAMP1(eBioscience,目录号:1D4B)
  6. 抗GM130(BD biosciences,目录号:35/GM130)
  7. 抗Rab5(Santa Cruz Biotechnologies,目录号:D-11)
  8. 抗钙联蛋白(Enzo Life Sciences,目录号:ADI-SPA-860)
  9. 抗COX IV(Cell Signaling Technology,目录号:4844)
  10. 抗大鼠Ig(H + L)-HRP(Jackson ImmunoResearch Laboratories,目录号:712-036-153)
  11. 抗小鼠Ig(H + L)-HRP(Jackson ImmunoResearch Laboratories,目录号:115-035-062)
  12. 抗兔Ig(H + L)-HRP(Jackson ImmunoResearch Laboratories,目录号:111-035-144)
  13. ECL底物(Thermo Fisher Scientific,目录号:34095)
  14. 蛋白酶抑制剂混合物(Thermo Fisher Scientific,目录号:87785)
  15. Halt Phosphatase Inhibitor Cocktail(Thermo Fisher Scientific,目录号:78420)
  16. 60%OptiPrep(Axis-Shield plc,目录号:1114542)
  17. 解决方案A(参见配方)
  18. 解决方案B(参见配方)
  19. 解决方案C(参见配方)
  20. 解决方案D(参见配方)
  21. 解决方案E(参见配方)
  22. 30%,23%,17%,11%和5%Optiprep(各2ml)(参见配方)
  23. TBS-T(参见配方)

设备

  1. 10厘米培养皿
  2. 离心管(Beckman Coulter,目录号:347357)
  3. 血细胞计数器
  4. 离心机(推荐使用冷却功能)
  5. 1 ml注射器,带29号针头

程序

当分析常规细胞系(例如 RAW264.7,Raji或HEK293T)或体积中具有大细胞溶质的细胞时应选择方案A,而应选择方案B用于小细胞或具有小细胞质,例如淋巴细胞。

  1. 从10cm皿(每个样品2个皿)中提取细胞的程序
    1. 层360μl的每种梯度溶液(30%,23%,17%,11% 5%OptiPrep)在离心管中用P200微量移液管,如图所示 在图1中

      图1.显示使用P200微量移液器分层梯度溶液的方法的示意图。 使用P200微量移液管(180μl×2次)将360μl每种梯度溶液小心倒入离心管中,不要产生气泡,也不要打扰其他层。在离心管中从最重(30%)到最轻(5%)溶液的层
    2. 将管在4°C下放置1小时以上,并保持静止直至使用。
    3. 确认细胞(例如 RAW264.7或Raji)是健康的,在显微镜下75-80%融合。
    4. (任选)用兴奋剂处理所需的时间(例如100ng/ml的LPS,持续30分钟)。
    5. 用10ml冰冷的PBS洗涤两次,转移到合适的管, 并用血细胞计数器和锥虫确定细胞密度和存活力   蓝色染色
    6. 将1.5〜2.0×10 7个细胞转移到新管中,并在4℃下以1,100rpm离心5分钟。
    7. 吸出上清液,轻拍管以松开细胞,并每管加入450μl冰冷的溶液E.
    8. 通过80号通过80-100通道匀浆细胞直到 细胞显示大约70-80%的台盼蓝阳性。 应保存细胞 冷。
    9. 在4℃下以1,000xg离心匀浆5分钟 并将整个上清液(核后级分)转移至新的 1.5ml管,并在1,000℃下在4℃下再次离心5分钟。 重复   这一步直到细胞碎片沉淀后才能看到 离心(通常3次) 注意:全部上清应该   取第二次离心,第三次离心 离心。 离心后上清液的初始体积 应该是大约400μl,并且在2次离心之后将是 减少到约。 350微升。
    10. 小心地将300μl上清液层叠在步骤A1(图3)中制备的梯度溶液的顶部
    11. 将离心管置于冷冻超速离心机转盘TLS-55中。
    12. 通过Optima MAX-TL在4℃下以39,000rpm(130,000xg)离心4小时。
      注意:将加速度(Accel)和减速度(Decel)分别设置为8和0(自由减速度)。
    13. 收获185微升每一部分,使用P200微量移液器制成11个部分,并转移到1.5毫升微管。
    14. 储存于-80℃直至使用。

  2. 小鼠原初B细胞的操作
    1. 层360μl的每种梯度溶液(30%,23%,17%,11% 5%OptiPrep)在离心管中用P200微量移液管
    2. 将管在4°C下放置1小时以上,并保持静止直至使用。
      通过使用CD43-微珠和MACS分离天然脾细胞 根据制造商的说明(请参见以下 链接: http://www.miltenyibiotec.co .jp /〜/media/Images/Products/Import/0001200/IM0001263.ashx )。
    3. (任选)用刺激剂处理1.5〜2.0×10 8个B细胞达期望的期间(例如,CpG-1668在100nM下1小时)。
    4. 用10ml冰冷PBS在合适的管中洗涤两次,和 用血细胞计数器和锥虫确定细胞密度和活力 蓝色染色
    5. 将细胞转移到新管中,并在4℃下以1,100rpm离心5分钟
    6. 吸出上清液,轻拍管以松开细胞,并每管加入450μl冰冷的溶液E.
    7. 通过大约200次通过29规格均匀化细胞 直到细胞显示70-80%的台盼蓝阳性。 细胞应该是 在冰上保持冷。
    8. 将匀浆在1,000×g离心5分钟 min,并在4℃下转移上清液(后核组分) 新的1.5 ml管。 重复此步骤三次。
    9. 小心地将300μl上清液层放在梯度顶部
    10. 将管置于冷冻超速离心机转子TLS-55中。
    11. 通过Optima MAX-TL在4℃以39,000rpm(130,000xg)离心4小时。
      注意:将加速度(Accel)和减速度(Decel)分别设置为8和0(自由减速度)。
    12. 使用P200微量吸管收集每个级分290μl以制备7个级分,并转移至1.5ml微量管。
    13. 储存于-80℃直至使用。

  3. 分馏的评价
    1. 取30μl的组分,并与等体积的2×SDS-PAGE样品缓冲液混合。
    2. 在95℃短暂变性后,应用15μl样品进行SDS-PAGE并转移到PVDF膜。
    3. 用封闭块封闭印迹,然后与孵育 针对细胞器标记物的一抗。 使用以下抗体 在这个协议; 抗LAMP1(稀释1:1,000)用于检测   晚期内体/溶酶体,抗GM130为顺式高尔基体,抗Rab5为 内体,ER的抗钙联蛋白和线粒体的抗COX IV, 分别
    4. 用TBS-T洗涤印迹3次以除去过量的抗体(5分钟×3次)
    5. 与与HRP偶联的正确二级抗体孵育 1小时[抗大鼠Ig(H + L)用于抗LAMP1,抗小鼠Ig(H + L)用于 抗GM130或抗-Rab5,抗兔Ig(H + L)用于抗钙联蛋白或COX IV。第二抗体的浓度大致为50  ng/ml]
    6. 用TBS-T洗涤印迹以除去过量的抗体(15分钟×3次)
    7. 与ECL底物孵育,并检测发光信号(图2)。

      图2.将小鼠幼稚B细胞的细胞内囊泡分级分离,并通过用针对以下细胞器标记物的特异性抗体的western印迹分析其分布;具有LAMP1的晚期内体/溶酶体,具有GM130的顺式高尔基体,具有Rab5的泛内含体,具有Calnexin的ER和具有COXIV的线粒体。。

笔记

  1. 对于细胞的均质化,可以使用Dounce匀浆器或其它装置,因为匀浆化困难程度取决于细胞类型。
  2. 对于诸如小鼠幼稚B细胞的小细胞,根据细胞密度,用29G注射器传代的次数可能不同。

食谱

  1. 解决方案A
    60%OptiPrep
  2. 解决方案B
    OptiPrep稀释剂
    (组件)
    100 ml
    (最终浓度)
    H sub 2 O
    〜42.8 ml

    1 M Hepes(pH7.5)
    15 ml
    150mM
    1 M KCl
    23.5毫升
    235 mM
    1 M MgCl 2
    1.2 ml
    12 mM
    1 M CaCl 2
    2.5 ml
    25 mM
    0.2 M EGTA
    15 ml
    30 mM
    用1M KOH调节至pH7.0,并加至100ml
  3. 解决方案C
    40%OptiPrep工作溶液
    2 vol。 的溶液A + 1vol。 的解决方案B
  4. 解决方案D
    工作溶液稀释剂
    (组分)100ml(最终浓度)
    (组件)
    100 ml
    (最终浓度)
    H sub 2 O
    〜81 ml

    1 M Hepes(pH7.5)
    5 ml
    50 mM
    1 M KCl
    7.8 ml
    78 mM
    1 M MgCl 2
    400μl
    4 mM
    1 M CaCl 2
    840微升
    8.4 mM
    0.2 M EGTA
    5 ml
    10 mM
    用1M KOH调节至pH7.0,并加至100ml
  5. 解决方案E
    均质介质
    (组件)
    100 ml
    (最终浓度)
    H sub 2 O
    〜81 ml

    1 M Hepes(pH7.5)
    5 ml
    50 mM
    1 M KCl
    7.8 ml
    78 mM
    1 M MgCl 2
    400μl
    4 mM
    1 M CaCl 2
    840微升
    8.4 mM
    0.2 M EGTA
    5 ml
    10 mM
    蔗糖(FW 342.30)
    8.56克
    250 mM
    用1M KOH调节至pH7.0,并加至100ml 使用前添加Halt蛋白酶抑制剂鸡尾酒和Halt磷酸酶抑制剂鸡尾酒
  6. 30%,23%,17%,11%和5%Optiprep(各2ml)
    (组件)
    30%
    23%
    17%
    11%
    5%
    溶液C(40%Optiprep)
    1500微升
    1150微升
    850微升
    550μl
    250微升
    解决方案D
    500微升
    850微升
    1150微升
    1450微升
    1750微升
    停止蛋白酶抑制剂*
    20微升
    20微升
    20微升
    20微升
    20微升
    停止磷酸酶抑制剂†
    20微升
    20微升
    20微升
    20微升
    20微升
    *停止蛋白酶抑制剂鸡尾酒
    †Halt磷酸酶抑制剂混合物


图3.超速离心之前完成的Opti-prep梯度图。

  1. TBS-T
    (组件)
    1 L
    (最终浓度)
    1M Tris-HCl(pH7.5)
    20ml
    20 mM
    NaCl
    8克
    137 mM
    吐温20
    1 ml
    0.1%

致谢

我们感谢K. Furuyama-Tanaka和S. Shimabukuro-Demoto的技术援助。 该协议改编自Axis-shield的密度梯度方案S44。 这项工作得到了下一代世界领先研究人员资助计划(下一个计划; NT-S。,LS134),日本教育,科学,体育和文化部的科学研究资助 对于NT-S,21390123,对于TK,25871165),以及来自国家全球健康和医学中心(用于NT-S,23S001)的赠款。

参考文献

  1. Kobayashi,T.,Shimabukuro-Demoto,S.,Yoshida-Sugitani,R.,Furuyama-Tanaka,K.,Karyu,H.,Sugiura,Y.,Shimizu,Y.,Hosaka,T.,Goto, ,Kato,N.,Okamura,T.,Suematsu,M.,Yokoyama,S.and Toyama-Sorimachi,N。(2014)。 组氨酸转运蛋白SLC15A4可协调mTOR依赖性炎症反应和致病性抗体产生。 免疫 41(3):375-388。
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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: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kobayashi, T., Tanaka, T. and Toyama-Sorimachi, N. (2015). Separation of Intracellular Vesicles for Immunoassays. Bio-protocol 5(16): e1571. DOI: 10.21769/BioProtoc.1571.
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