搜索

VAMP8-3xHA Uptake Assay in HeLa Cells
HeLa 细胞中的VAMP8-3xHA摄取试验   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Transmembrane proteins are rarely exclusively localized to a specific vesicle or an organelle. Most transmembrane proteins undergo complicated trafficking routes. Thus, transmembrane proteins are under constant flux, and at steady state, found on a variety of vesicles or organelles. This characteristic makes the study of their trafficking routes complex, since at any given moment, different molecules are often being trafficked in opposing directions. Pulse-chase experiments can temporally track a specific pool of a transmembrane protein of interest, allowing for the kinetic description of its trafficking route. This type of technique has been used extensively to follow a large array of plasma membrane localized proteins (Diril et al., 2006; Jean et al., 2010). Here, we describe a method that allows the study of VAMP8 trafficking from the plasma membrane to endolysosomal compartments. This method was used to describe a role for MTMR13 and RAB21 in the regulation of VAMP8 trafficking to endolysosomes (Jean et al., 2015).

Keywords: Membrane trafficking(膜转运), Endosomal sorting(胞内分选), Autophagy(自噬)

Materials and Reagents

  1. Costar® 24 Well Clear TC-Treated Well Plates (Corning, catalog number: 3526 ) 

  2. #1.5 round glass coverslip (Ted Pella, catalog number: 260368 ) 

  3. 100 mm Tissue Culture Dish (Corning, catalog number: 430167 ) 

  4. 0.22 µm filtering unit (Genesee Scientific Corporation, catalog number: 25-227 )
  5. HeLa cells (ATCC, catalog number: CCL-2 ) 

  6. JetPRIME (Polyplus-transfection, catalog number: 114-07 ) 

  7. pCDNA3-VAMP8-3xHA expression plasmid (self-made) (Jean et al., 2015)
  8. Dulbecco’s Modified Eagle Medium with High glucose with 4 mM L-Glutamine and sodium pyruvate (GE Healthcare, HycloneTM, catalog number: SH30243.FS ) (see Recipes for complete DMEM) 

  9. Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml ) 

  10. Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  11. Trypsin-EDTA (0.25%) (Life Technologies, catalog number: 25200-056 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
  12. 0.4% Trypan-Blue (Life Technologies, catalog number: 15250-061 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
  13. Rabbit anti-HA antibody (Abcam, catalog number: ab9110 ) 

  14. Earl’s Balanced Salt Solution (with sodium bicarbonate, without phenol red, liquid, sterile- filtered) (Sigma-Aldrich, catalog number: E3024-500 ml ) 

  15. Bovine Serum Albumin (Fraction V, Heat shock treated) (Thermo Fisher Scientific, catalog number: BP1600-100 ) 

  16. Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S671-3 )

  17. Potassium Chloride (KCl) (VWR International, catalog number: BDH9258-500 g ) 

  18. Sodium Phosphate Dibasic Anhydrous (Na2HPO4) (Thermo Fisher Scientific, catalog number: S374500 )

  19. Potassium Phosphate Monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: P285-S500 ) 

  20. Paraformaldehyde (Thermo Fisher Scientific, catalog number: AC41678-5000 ) 

  21. Goat Serum (Life Technologies, catalog number: 16210-064 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 16210-064”.
  22. Triton X-100 (Sigma-Aldrich, catalog number: X100-500 ml ) 

  23. Mouse anti-human Lamp1 (Developmental Studies Hybridoma Bank, catalog number: H4A3 ) 

  24. Mouse anti-EEA1 (BD Biosciences, catalog number: 610457 ) 

  25. Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Life Technologies, catalog number: A-11029 ) 

    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11029”.
  26. Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 546 conjugate (Life Technologies, catalog number: A11035 ) 

    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: A11035”.
  27. [4’, 6-Diamidino-2-Phenylindole, Dihydrochloride] (DAPI) (Life Technologies, catalog number: D1306 ) 

    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306”.
  28. FluorSave reagent (Merck Millipore Corporation, Calbiochem®, catalog number: 345789 ) 

  29. Complete DMEM (see Recipes) 

  30. 1x Phosphate Buffer Saline (PBS) (see Recipes) 

  31. 4% Paraformaldehyde solution (see Recipes) 

  32. Blocking buffer (see Recipes) 

  33. Antibody incubation buffer (see Recipes) 


Equipment

  1. 37 °C, 5% CO2 cell culture incubator [similar to Thermo Scientific Heracell VIOS 160i CO2 incubator (Thermo Fisher Scientific, catalog number: 51030285 )]

  2. Tissue culture biosafety cabinet [similar to Thermo Scientific 1300 Series Class II (Thermo Fisher Scientific, catalog number: 1323)] 

  3. Hemacytometer (VWR International, catalog number: 100498-470 ) 

  4. Pipettes 10 μl, 100 μl and 1,000 μl (Eppendorf, catalog numbers: 3120000020 , 3120000046 and 31200000623 )
  5. Vacuum Flask (self-made)
    Note: Required to remove media from the 24 well plates during the immunofluorescence protocol.
  6. Microcentrifuge (Eppendorf, catalog number: 5415D ) 


Procedure

  1. Day 1: Seeding cells
    1. Add 1 circular sterile coverglass per well of a 24 well plate. Use sterile forceps to manipulate cover glasses. (The number of wells used depends on the number of samples and time points). Coverglasses are sterilized by autoclaving in a glass dish for 30 min.
    2. Remove media from 100 mm stock HeLa plate, and wash with 5 ml of 1x PBS. Either room temperature or 37 °C pre-warmed PBS is suitable for the steps listed in procedure A.
    3. Remove PBS, add 1 ml of 0.25% trypsin-EDTA, and incubate at 37 °C for 3 min. 

    4. Wash cells off the plate with 10 ml of complete DMEM. 

    5. Pipet 20 μl of the cell suspension, and add to 100 μl of Trypan-Blue solution in an Eppendorf tube. 

    6. Pipet 10 μl of this cell/Trypan-Blue dilution into a hemacytometer chamber. 

    7. Count viable cells using the hemacytometer on a standard microscope. Viable cells will be Trypan-Blue negative. 

    8. Add a total of 1.5 x 104 cells per well of a 24-well plate. 

    9. Bring to 1.5 ml of complete DMEM per well, and move to 37 °C incubator overnight (o/n). 


  2. Day 2: HeLa cells Transfection using JetPrime
    1. Remove media from each well and replace with 0.5 ml of complete DMEM. 

    2. In a 1.5 ml Eppendorf tube, add 500 ng of pCDNA3-VAMP8-3xHA and adjust to a final volume of 100 μl with JetPrime buffer. The amount of DNA, JetPrime buffer and JetPrime mentioned are sufficient for the transfection of 6 wells of a 24 well plate. This can be scaled up or down depending on the number of wells needed.
    3. Vortex for 10 sec, and spin down in a microcentrifuge at 1,000 rpm for 5 sec. 

    4. Add 2 μl of JetPrime transfection reagent. 

    5. Vortex for 10 sec, and spin down as in step B3. 

    6. Incubate at room temperature for at least 10 min. 

    7. Add 15 μl of transfection mix per well. Each master mix can be used on 6 wells.
    8. Incubate 6 hours at 37 °C in the tissue culture incubator. 

    9. Remove the media and replace with 1.5 ml of complete DMEM. 

    10. Incubate overnight in the 37 °C tissue culture incubator. 


  3. Day 3: VAMP8 endocytic uptake assay
    1. Wash transfected cells with 1 ml of ice-cold DMEM. Repeat one more time for a total of two washes. Remove all the media. 

    2. Dilute rabbit anti-HA antibody in ice cold complete DMEM to a final dilution of 1:200. 

    3. Add 150 μl of diluted anti-HA antibody to each well. 

    4. Incubate on ice for 60 min with occasional rocking. (This step allows binding of the antibody 
to plasma membrane-localized VAMP8). 

    5. Wash cells three times with 1ml of ice-cold complete DMEM. (This step removes all unbound antibodies). 

    6. Wash cells two times with 1 ml of pre-warmed EBSS (37 °C). 

    7. Add 1 ml of pre-warmed EBSS to each well, and incubate in the 37 °C tissue culture incubator 
for 15, 45 and 90 min. 
Please refer to the section, 'Important experimental controls for uptake assay validation,' in order to prepare all the necessary controls.

  4. Immunofluorescence (Day 3 and 4)
    1. At each time point, remove the appropriate coverglass and transfer into a new well of a new 24-well plate containing 1 ml of 1x PBS. 

    2. Remove the 1x PBS, and replace with 1 ml of the 4% paraformaldehyde solution. 

    3. Incubate for 15 min at room temperature. 

    4. Remove 4% paraformaldehyde solution, and wash three times with 1 ml of 1x PBS. For each wash, incubate 5 min at room temperature. 

    5. Remove last wash and block in 1 ml of blocking buffer for 1 h at room temperature. 

    6. Dilute mouse anti-Lamp1 or mouse anti-EEA1 to final dilutions of 1:100 or 1:1,000, respectively, in antibody incubation buffer. 

    7. Remove blocking buffer and replace with 100 μl of the appropriately diluted antibody, and incubate at 4 °C o/n. 

    8. Remove diluted antibody solution, and wash 3 times with 1 ml of 1x PBS. For each wash, incubate 5 min at room temperature. 

    9. Dilute Alexa-coupled secondary antibodies. Dilute goat anti-mouse Alexa-488 and goat anti-rabbit Alexa-546 to a final dilution of 1:250 in antibody dilution buffer. 

    10. Remove wash solution, add 200 μl of diluted secondary antibody solution, and incubate 1 h
at room temperature in the dark to minimize photobleaching. 

    11. Remove secondary antibody-containing solution, and wash three times with 1 ml of 1x PBS. 
For each wash, incubate 5 min at room temperature. 

    12. Add 200 μl of 1x PBS containing 1 μg/ml of DAPI and incubate 5 min at room temperature.

    13. Remove DAPI-containing solution and wash twice with 1 ml of 1x PBS. For each wash, incubate 5 min at room temperature. 

    14. Pipet 10 μl of FluorSave mounting media on a microscope slide. Remove coverglass from individual wells using forceps, and gently remove as much liquid as possible. Gently drop the coverglass onto the slide and the mounting media, making sure that no air bubbles are trapped between the coverglass and the slide. Remove extra mounting solution, and let the slide cure o/n at 4 °C. 

    15. Image on an appropriate confocal or widefield microscopy system. 


  5. Automated quantification of colocalization
    1. Export microscope images to 16 bit tiff files.
    2. Use CellProfiler software to analyze colocalization (http://www.cellprofiler.org).
    3. Using CellProfiler, generate a pipeline that allows the identification of cell boundaries and of the different objects: EEA1 vesicles, LAMP1 vesicles and VAMP8 vesicles. See Jean et al. (2015) for detailed methods.
    4. Measure object colocalization by following the recommendations found at http://www.cellprofiler.org/linked_files/ExampleColocalization_Tutorial.pdf).
    5. Once the number of colocalized objects is established, correlate the percentage of colocalized objects per cells using excel.

    Important experimental controls for uptake assay validation
    In order to insure valid and interpretable endocytic uptake experiments, the following controls should be performed:
    Validate that the antibody labels only surface localized proteins at time 0.
    1. Include a condition that is held only at 4 °C for the antibody binding step but not permitted to undergo uptake at 37 °C. Perform the immunofluorescence as described for other timepoints with uptake conditions. Staining should only be localized around the plasma membrane, which will be evident when analyzed by 
confocal microscopy.
    2. Given that most uptake experiments rely on transient transfection and detection of an epitope tag, a mixed population of cells will be present in the dish. Thus, in line with transfection efficiency, untransfected cells should not display any staining. 

    3. Perform a 4 °C incubation in the absence of chase antibody (HA antibody used to label VAMP8:3xHA) and fix the cells. Stain cells using a conventional immunofluorescence protocol (with primary HA antibody and secondary antibodies Goat anti-rabbit Alexa-546) to identify the steady-state localization of the protein of interest. For many proteins, the total protein staining at steady-state typically detects signal mainly on internal punctae, reflecting protein trafficking through internal compartments. 

      In conclusion, the lack of plasma membrane staining in untransfected cells together with exclusive plasma membrane staining in transfected cells at time 0, coupled with a punctate pattern in permeabilized cells, argues for staining specificity. Rapid endocytosis of chased proteins (VAMP8:3xHA) further indicates a functional uptake assay. Altogether, these results validate the endocytic uptake assay. Note that this assay requires additional modifications to monitor recycling of surface-labeled cargo to the plasma membrane (van Weert et al., 2000).


      Figure 1. Control experiments to insure a functional uptake assay. A. Cells labeled with anti-HA antibody and without a 37 °C incubation show only surface membrane staining, while (B) cells chased at 37 °C for 30 min show intracellular staining. C. Total VAMP8:3xHA detected by regular immunofluorescence shows mostly an intracellular pattern.

Recipes

  1. Complete DMEM

    Remove 55 ml of DMEM from the purchased 500 ml HyClone bottle
    Add 50 ml of fetal bovine serum (yields a final concentration of 10%)
    Add 5 ml of Penicillin/Streptomycin solution (yields a final concentration of 1%)
    Filter sterilized with filtering device (0.2 μm)
  2. 1x phosphate buffer saline (PBS)

    Mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4
    Add 900 ml of dH2O
    Adjust pH to 7.4
    Adjust final volume to 1,000 ml
    Sterilized by autoclaving at 121 °C for 20 min
  3. 4% paraformaldehyde solution

    Add 4 g of paraformaldehyde to 80 ml of 1x PBS
    Heat to 65 °C (in a water bath) with occasional vortexing
    Adjust pH to 7.4 with NaOH
    Complete to 100 ml with 1x PBS
    Filter sterilize (0.2 μm)
    Use fresh
  4. Blocking buffer
    Mix 5 ml of Goat serum to 95 ml of 1x PBS (yields a final concentration of 5%)
    Add 0.3 ml of Triton X-100 (Use a wide bore tip to pipette Triton X-100) (yields a final concentration of 0.3%)
    Mix well
  5. Antibody incubation buffer

    Add 0.1 g of BSA to 10 ml of 1x PBS (yields a final concentration of 1%)
    Add 0.03 ml of Triton X-100 (Use a wide bore tip to pipette Triton X-100) (yields a final concentration of 0.3%)
    Mix well

Acknowledgments

The uptake assay was adapted from the previously published study (Miller et al., 2011) and was performed in (Jean et al., 2015). The immunofluorescence protocol was adapted from Cell Signaling Technology, http://www.cellsignal.com/common/content/content.jsp?id=if. This work was supported by FRSQ, AHA and CRS postdoctoral fellowships to SJ, and NIH RO1 GM078176 and support from the SDCSB NIH P50 GM085764 to AAK.

References

  1. Diril, M. K., Wienisch, M., Jung, N., Klingauf, J. and Haucke, V. (2006). Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling. Dev Cell 10(2): 233-244.
  2. Jean, S., Cox, S., Nassari, S. and Kiger, A. A. (2015). Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion. EMBO Rep 16(3): 297-311.
  3. Jean, S., Mikryukov, A., Tremblay, M. G., Baril, J., Guillou, F., Bellenfant, S. and Moss, T. (2010). Extended-synaptotagmin-2 mediates FGF receptor endocytosis and ERK activation in vivo. Dev Cell 19(3): 426-439.
  4. Miller, S. E., Sahlender, D. A., Graham, S. C., Honing, S., Robinson, M. S., Peden, A. A. and Owen, D. J. (2011). The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM. Cell 147(5): 1118-1131.
  5. van Weert, A. W., Geuze, H. J., Groothuis, B. and Stoorvogel, W. (2000). Primaquine interferes with membrane recycling from endosomes to the plasma membrane through a direct interaction with endosomes which does not involve neutralisation of endosomal pH nor osmotic swelling of endosomes. Eur J Cell Biol 79(6): 394-399.

简介

跨膜蛋白很少专门定位于特定的囊泡或细胞器。大多数跨膜蛋白经历复杂的运输路线。因此,跨膜蛋白在恒定通量下,并在稳定状态,发现在各种囊泡或细胞器。这个特征使得他们的贩运路线的研究复杂,因为在任何给定时刻,不同的分子通常在相反的方向上被贩运。脉冲追踪实验可以暂时跟踪感兴趣的跨膜蛋白的特定池,允许其运输路线的动力学描述。这种类型的技术已广泛用于跟踪大量的质膜定位蛋白质(Diril等人,2006; Jean等人,2010)。在这里,我们描述了一种方法,允许研究VAMP8贩运从质膜到内溶酶体隔室。该方法用于描述 MTMR13 和 RAB21 在调节VAMP8向内溶酶体的运输中的作用(Jean等人,,2015)。

关键字:膜转运, 胞内分选, 自噬

材料和试剂

  1. Costar ? 24透明TC处理孔板(Corning,目录号:3526)
  2. #1.5圆玻璃盖玻片(Ted Pella,目录号:260368)
  3. 100mm组织培养皿(Corning,目录号:430167)
  4. 0.22μm过滤单元(Genesee Scientific Corporation,目录号:25-227)
  5. HeLa细胞(ATCC,目录号:CCL-2)
  6. JetPRIME(Polyplus转染,目录号:114-07)
  7. pCDNA3-VAMP8-3xHA表达质粒(自制)(Jean ,2015)
  8. 具有高葡萄糖与4mM L-谷氨酰胺和丙酮酸钠的Dulbecco改良的Eagle培养基(GE Healthcare,Hyclone TM ,目录号:SH30243.FS)(参见完全DMEM的配方)
  9. 胎牛血清(Sigma-Aldrich,目录号:F2442-500ml)
  10. 青霉素 - 链霉素溶液(Life Technologies,目录号:15140-122)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140-122"
  11. 胰蛋白酶-EDTA(0.25%)(Life Technologies,目录号:25200-056)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25200-056"
  12. 0.4%台盼蓝(Life Technologies,目录号:15250-061) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15250-061"
  13. 兔抗HA抗体(Abcam,目录号:ab9110)
  14. Earl's平衡盐溶液(用碳酸氢钠,不含酚红,液体,无菌过滤)(Sigma-Aldrich,目录号:E3024-500ml)
  15. 牛血清白蛋白(级分V,热休克处理)(Thermo Fisher Scientific,目录号:BP1600-100)
  16. 氯化钠(NaCl)(Thermo Fisher Scientific,目录号:S671-3)
  17. 氯化钾(KCl)(VWR International,目录号:BDH9258-500g)
  18. 磷酸氢二钠无水(Na 2 HPO 4)(Thermo Fisher Scientific,目录号:S374500)
  19. 磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,目录号:P285-S500)
  20. 多聚甲醛(Thermo Fisher Scientific,目录号:AC41678-5000)
  21. 山羊精华(Life Technologies,目录号:16210-064)
    注意:目前,"赛默飞世尔科技,Gibco TM ,目录号:16210-064"
  22. Triton X-100(Sigma-Aldrich,目录号:X100-500ml)
  23. 小鼠抗人类Lamp1(Developmental Studies Hybridoma Bank,目录号:H4A3)
  24. 小鼠抗EEA1(BD Biosciences,目录号:610457)
  25. 山羊抗小鼠IgG(H + L)二抗,Alexa Fluor <488缀合物(Life Technologies,目录号:A-11029)
    注意:目前,"Thermo Fisher Scientific,Invitrogen TM
  26. 山羊抗兔IgG(H + L)第二抗体,Alexa Fluor 546缀合物(Life Technologies,目录号:A11035)
    注意:目前,"Thermo Fisher Scientific,Invitrogen />
  27. [4',6-二脒基-2-苯基吲哚,二盐酸盐](DAPI)(Life Technologies,目录号:D1306)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:D1306" br />
  28. FluorSave试剂(Merck Millipore Corporation,Calbiochem ,目录号:345789)
  29. 完成DMEM(参见配方)
  30. 1x磷酸盐缓冲盐水(PBS)(参见配方)
  31. 4%多聚甲醛溶液(参见配方)
  32. 阻止缓冲区(参见配方)
  33. 抗体孵育缓冲液(参见配方)

设备

  1. 37℃,5%CO 2细胞培养箱[类似于Thermo Scientific Heracell VIOS 160i CO 2培养箱(Thermo Fisher Scientific,目录号:51030285)]。 >
  2. 组织培养生物安全柜[类似于Thermo Scientific 1300 Series Class II(Thermo Fisher Scientific,目录号:1323)]
  3. 血细胞计数器(VWR International,目录号:100498-470)
  4. 移液管10μl,100μl和1000μl(Eppendorf,目录号:3120000020,312000046和31200000623)
  5. 真空瓶(自制)
    注意:在免疫荧光方案期间需要从24孔板中去除培养基。
  6. 微量离心机(Eppendorf,目录号:5415D)

程序

  1. 第1天:播种细胞
    1. 在24孔板的每个孔中加入1个圆形无菌盖玻片。使用 无菌镊子操纵封面眼镜。 (使用的井数 取决于样品的数量和时间点)。封面是 在玻璃皿中高压灭菌30分钟
    2. 去掉 来自100mm股票HeLa板的培养基,并用5ml 1x PBS洗涤。或者 ?室温或37℃预热的PBS适合于这些步骤 列在程序A中。
    3. 取出PBS,加入1ml 0.25%胰蛋白酶-EDTA,37℃孵育3分钟。
    4. 用10ml完全DMEM洗涤板上的细胞。
    5. 吸取20微升的细胞悬液,并加入到100微升的锥虫蓝溶液在Eppendorf管中。
    6. 吸取10微升的这个单元格/台盼蓝稀释到血细胞计数室。
    7. 在标准显微镜上使用血细胞计数器计数活细胞。活细胞将是台盼蓝阴性。
    8. 在24孔板的每孔中加入总共1.5×10 4个细胞。
    9. 每孔加入1.5ml完全DMEM,并移至37℃培养箱过夜(o/n)。

  2. 第2天:HeLa细胞使用JetPrime转染
    1. 从每个孔中删除培养基,并更换为0.5毫升的完整DMEM。
    2. 在1.5ml Eppendorf管中,加入500ng的pCDNA3-VAMP8-3xHA和 用JetPrime缓冲液调节至终体积为100μl。大量的 DNA,JetPrime缓冲区和JetPrime就足够了 转染24孔板的6个孔。这可以按比例放大或 取决于所需的井数
    3. 涡旋10秒,并在微量离心机中以1,000rpm离心5秒。
    4. 加入2μl的JetPrime转染试剂。
    5. 涡旋10秒,并如步骤B3中旋转。
    6. 在室温下孵育至少10分钟。
    7. 每孔加入15μl转染混合物。每个主混合物可用于6口井
    8. 在37℃下在组织培养箱中孵育6小时。
    9. 取出介质,并更换为1.5毫升完整的DMEM。
    10. 在37℃组织培养箱中孵育过夜。

  3. 第3天:VAMP8内吞摄取测定
    1. 用1ml冰冷的DMEM洗涤转染的细胞。重复一次,共两次洗涤。删除所有介质。
    2. 在冰冷的完全DMEM中稀释兔抗HA抗体至1:200的最终稀释度。
    3. 向每个孔中加入150μl稀释的抗-HA抗体。
    4. 在冰上孵育60分钟,偶尔摇摆。 (这一步 允许抗体与质膜定位的VAMP8的结合)。
    5. 用1ml冰冷的完全DMEM洗涤细胞三次。 (该步骤除去所有未结合的抗体)。
    6. 用1ml预热的EBSS(37℃)洗涤细胞两次。
    7. 向每个孔中加入1ml预热的EBSS,并在37℃下孵育 组织培养孵育15,45和90分钟。请参考 section,'重要的实验控制摄取测定验证, 以便准备所有必要的控制。

  4. 免疫荧光(第3天和第4天)
    1. 在每个时间点,取出适当的盖玻片并转移到 ?新的含有1ml 1×PBS的24孔板的新孔。
    2. 取出1×PBS,并更换1毫升的4%多聚甲醛溶液。
    3. 在室温下孵育15分钟。
    4. 除去4%多聚甲醛溶液,用1 ml洗涤三次 的1x PBS。对于每次洗涤,在室温下孵育5分钟。
    5. 取出最后一次洗涤并在1ml封闭缓冲液中室温封闭1小时。
    6. 稀释小鼠抗Lamp1或小鼠抗EEA1到的最终稀释度 在抗体孵育缓冲液中分别为1:100或1:1,000。
    7. 除去封闭缓冲液,更换100微升适当的稀释抗体,并在4°C o/n孵育。
    8. 取出稀释的抗体溶液,并用1ml 1×PBS洗涤3次。对于每次洗涤,在室温下孵育5分钟。
    9. 稀释Alexa偶联的二抗。稀释山羊抗小鼠 Alexa-488和山羊抗兔Alexa-546至1:250的最终稀释度 ?抗体稀释缓冲液。
    10. 取出洗涤液,加入200μl ?稀释的二抗溶液,并孵育1个帽室 温度在黑暗中,以尽量减少光漂白。
    11. 去掉 第二抗体溶液,并用1ml洗涤3次 的1x PBS。对于每次洗涤,在室温下孵育5分钟。
    12. 加入200微升含有1微克/毫升DAPI的1×PBS,并在室温下孵育5分钟。
    13. 除去含DAPI的溶液,并用1ml 1×PBS洗涤两次。对于每次洗涤,在室温下孵育5分钟。
    14. 吸取10微升的FluorSave安装在显微镜载玻片上。 使用镊子从个别井中删除盖玻片,轻轻删除 ?尽可能多的液体。将盖玻片轻轻放在幻灯片上 和安装介质,确保没有气泡被捕获 在盖玻片和滑块之间。移除额外的安装解决方案, 并使载玻片在4℃下固化o/n。
    15. 在适当的共聚焦或宽视场显微镜系统上的图像。

  5. 自定量化共定位
    1. 将显微镜图像导出到16位tiff文件
    2. 使用CellProfiler软件分析共定位( http://www.cellprofiler.org )。
    3. 使用CellProfiler,生成一个管道,允许 识别细胞边界和不同的对象:EEA1 囊泡,LAMP1囊泡和VAMP8囊泡。见Jean et al。(2015)for 详细方法
    4. 通过跟随测量对象共定位 在 http://www.cellprofiler.org/linked_files/ExampleColocalization_Tutorial.pdf 中找到的建议) 。
    5. 一旦建立了共定位对象的数量,就关联 ?使用excel的每单元colocalized对象的百分比。

    摄取测定验证的重要实验性控制
    为了确保有效和可解释的内吞摄取实验,应进行以下控制:
    验证抗体在时间0时仅标记表面定位的蛋白质。
    1. 包括仅在4℃下保持抗体结合的条件 步骤,但不允许在37℃摄取。执行 如其它具有摄取的时间点所述 条件。染色应仅在等离子体周围定位 膜,当通过共聚焦显微镜分析时,这将是明显的。
    2. 鉴于大多数摄取实验依赖于瞬时转染 并检测表位标签,混合细胞群将会 存在于菜。因此,与转染效率一致, 未转染的细胞不应显示任何染色。
    3. 执行4 ?在不存在追踪抗体的情况下孵育(使用HA抗体 标记VAMP8:3xHA)并固定细胞。使用常规染色细胞 免疫荧光方案(具有原发性HA抗体和继发性抗体) 抗体山羊抗兔Alexa-546)以鉴定稳态 感兴趣的蛋白质的定位。对于许多蛋白质,总 蛋白染色在稳态典型地检测信号主要开 内部斑点,反映通过内部的蛋白质贩运 隔间。
      总之,缺乏质膜染色 在未转染的细胞中与专有的质膜染色 在转染的细胞中在时间0,与点状图案偶联 透化细胞,争论染色特异性。快速内吞 的追踪蛋白(VAMP8:3xHA)进一步表明功能摄取 测定。总之,这些结果验证内吞摄取测定。 注意,该测定需要额外的修饰来监测 将表面标记的货物再循环至质膜(van Weert等人, al。,2000)。


      图1.确保功能的控制实验 吸收测定。A.用抗HA抗体标记的细胞,无37℃ ?孵化只显示表面膜染色,而(B)细胞追踪 在37℃下30分钟显示细胞内染色。 C.总VAMP8:3xHA 通过常规免疫荧光检测显示大部分细胞内 模式。

食谱

  1. 完成DMEM
    从购买的500 ml HyClone瓶中取出55 ml DMEM
    加入50ml胎牛血清(最终浓度为10%) 加入5ml青霉素/链霉素溶液(得到最终浓度为1%) 用过滤装置(0.2μm)过滤灭菌
  2. 1×磷酸盐缓冲盐水(PBS)
    将8g的NaCl,0.2g的KCl,1.44g的Na 2 HPO 4和0.24g的KH 2 PO 4
    加入900ml dH 2 O
    将pH调节至7.4
    将最终体积调整为1,000 ml
    在121℃高压灭菌20分钟,灭菌处理
  3. 4%多聚甲醛溶液
    向80 ml 1x PBS中加入4 g多聚甲醛
    加热至65°C(在水浴中),偶尔涡旋
    用NaOH调节pH至7.4 用1x PBS完成至100 ml
    过滤灭菌(0.2μm)
    使用新鲜的
  4. 阻塞缓冲区
    将5毫升山羊血清与95毫升1×PBS(产生终浓度5%)混合 加入0.3ml Triton X-100(使用大口径吸头Triton X-100)(最终浓度为0.3%)
    混合良好
  5. 抗体温育缓冲液
    将0.1g BSA加入10ml 1×PBS(产生最终浓度为1%)中 加入0.03ml Triton X-100(使用大口径吸头Triton X-100)(最终浓度为0.3%)
    混合良好

致谢

摄取测定改变自先前公开的研究(Miller等人,2011),并且在(Jean等人,2015)中进行。免疫荧光方案改编自Cell Signaling Technology, http://www.cellsignal .com/common/content/content.jsp?id = if 。这项工作是由FRSQ,AHA和CRS博士后研究支持SJ和NIH RO1 GM078176和从SDCSB NIH P50 GM085764到AAK的支持。

参考文献

  1. Diril,M.K.,Wienisch,M.,Jung,N.,Klingauf,J.and Haucke,V。(2006)。 Stonin 2是一种用于突触结合蛋白内化和回收的AP-2依赖性内吞分选适配子。 Dev Cell 10(2):233-244。
  2. Jean,S.,Cox,S.,Nassari,S.and Kiger,A.A。(2015)。 饥饿诱导的MTMR13和RAB21活性调节VAMP8以促进自噬体 - 溶酶体融合。 em> EMBO Rep 16(3):297-311。
  3. Jean,S.,Mikryukov,A.,Tremblay,M.G.,Baril,J.,Guillou,F.,Bellenfant,S.and Moss,T。 Extended-synaptotagmin-2在体内介导FGF受体内吞作用和ERK活化 。 Dev Cell 19(3):426-439。
  4. Miller,S.E.,Sahlender,D.A.,Graham,S.C.,Honing,S.,Robinson,M.S.,Peden,A.A。和Owen,D.J。(2011)。 网格蛋白适配子CALM对小R-SNARE的内吞作用的分子基础。 147(5):1118-1131。
  5. van Weert,A.W.,Geuze,H.J.,Groothuis,B。和Stoorvogel,W。(2000)。 Primaquine通过与内体的直接相互作用干扰从内体到质膜的膜再循环,其不涉及中和核内体pH或核内体的渗透性肿胀。 Eur J Cell Biol 79(6):394-399。
  • English
  • 中文翻译
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: © 2016 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. Jean, S. and Kiger, A. A. (2016). VAMP8-3xHA Uptake Assay in HeLa Cells. Bio-protocol 6(4): e1739. DOI: 10.21769/BioProtoc.1739.
  2. Jean, S., Cox, S., Nassari, S. and Kiger, A. A. (2015). Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion. EMBO Rep 16(3): 297-311.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。