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Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells
通过抗体喂养追踪活体极化MDCK细胞中表位标记的 Syntaxin 3的胞吞作用和细胞内转运   

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Molecular Biology of the Cell
Oct 2017

Abstract

The uptake and trafficking of cell surface receptors can be monitored by a technique called ‘antibody-feeding’ which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.

Keywords: Antibody-feeding assay (抗体喂养试验), Syntaxin (突触融合蛋白), SNARE (SNARE), Internalization (内化), Apical-basolateral polarity (顶端 - 基底外侧极性), Epithelial cells (上皮细胞), MDCK cells (MDCK细胞)

Background

The SNARE protein Syntaxin 3 (Stx3) is known to establish apical-basolateral polarity in polarized epithelial cells (Low et al., 1996; Delgrossi et al., 1997; Weimbs et al., 1997; Low et al., 1998; Li et al., 2002; Low et al., 2006). Apical localization of Stx3 depends on a conserved targeting motif near the N-terminus of the protein (ter Beest et al., 2005; Sharma et al., 2006). A fraction of Stx3 is also known to localize to late endosomes/lysosomes in Madin-Darby Canine Kidney (MDCK) cells, which form tight-junctions, establish apical and basolateral polarity, and adopt a columnar morphology when grown to confluence. To determine the origin of this population of Stx3 and to investigate its endosomal trafficking in polarized MDCK cells, we designed an ‘antibody-feeding assay’ protocol. Using this assay and other experiments, we have shown that Stx3 is ubiquitinated at lysine residues in a basic juxtamembrane region and that ubiquitination facilitates the endocytosis of Stx3 from the basolateral membrane leading to trafficking to intraluminal vesicles of multivesicular bodies, and eventually secretion with exosomes (Giovannone et al., 2017). A non-ubiquitinatable mutant (Stx3-5R) exhibits decreased endosomal trafficking and exosomal secretion (Giovannone et al., 2017). Using an antibody-feeding assay, we can monitor where Stx3 is trafficked after being delivered to the apical or basolateral membrane. A cassette containing two Myc epitope tags and one hexa-histidine tag was added to the C-terminus of Stx3 by molecular cloning. These tags are exposed to the extracytoplasmic side of the plasma membrane when tagged Stx3 is present on the surface in transfected cells. We have previously shown that these C-terminal Myc2-His6 tags are accessible to the 9E10 anti-c-Myc monoclonal antibody and do not interfere with the known surface polarity of several syntaxins (Low et al., 2000; Kreitzer et al., 2003; Low et al., 2006; Sharma et al., 2006; Reales et al., 2011; Giovannone et al., 2017). Epitope-tagged Stx3 is stably expressed in MDCK cells using a tetracycline-controlled transcriptional activation system which allows using uninduced cells as a negative control. Cells are cultured on Transwell permeable membranes until fully polarized (Figure 1), incubated with anti-c-Myc antibody, and harvested for analysis by immunofluorescence microscopy at various time points. Cells were also incubated with anti-M6PR antibody (late endosomal marker). Lastly, cells were stained with DAPI and secondary antibodies against the 9E10 anti-c-Myc monoclonal antibody (for Stx3) and anti-M6PR antibody.


Figure 1. Schematic drawing of a Transwell polycarbonate membrane cell culture insert inside a well of a typical 12-well cell culture dish. Cells are cultured on top of the membrane and will form a tight monolayer that seals off the polycarbonate membrane thereby separating the apical media compartment from the basolateral media compartment.

Materials and Reagents

  1. 10 cm cell culture dishes (Corning, Falcon®, catalog number: 353003 )
  2. 10 ml serological pipette (VWR, catalog number: 89130-898 )
  3. 50 ml conical tubes (Corning, catalog number: 430290 )
  4. 12-well cell culture plates with 0.4 µm pore polycarbonate Transwell supports (Corning, catalog number: 3401 )
  5. 12-well cell culture plates (Corning, Costar®, catalog number: 3512 )
  6. Razor blades (VWR, catalog number: 55411-050 )
  7. Paper towels
  8. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34120 )
  9. Parafilm (BEMIS, catalog number: PM996 )
  10. Plastic pencil box with lid (VWR, catalog number: 500003-109 )
    Manufacturer: Janitorial Supplies, catalog number: AVT34104 .
  11. Microscope slides (any standard slides from any supplier will be fine)
  12. Micro cover glasses, 18 x 18 mm, No. 1 thickness is 0.13 to 0.17 mm (VWR, catalog number: 48366-045 )
  13. Madin-Darby Canine Kidney (MDCK) cells stably expressing C-terminally Myc2-His6-tagged Stx3
    Note: Madin-Darby Canine Kidney (MDCK) cells stably expressing C-terminally Myc2-His6-tagged Stx3 using a doxycycline-inducible expression system have been described in detail in Sharma et al., 2006. The parental cell line, expressing the TET transactivator is required to produce doxycycline-inducible, stably transfected cells for one’s gene of interest and have been generated in the laboratory of the senior author (TW). Cells may be requested from the authors. Original MDCK cells and several subclones are also available from ATCC (ATCC, catalog number: CCL-34 and others) but these cells may differ in some characteristics from those used here.
  14. Sterile 1x Dulbecco’s phosphate buffered saline (DPBS) without calcium or magnesium (Mediatech, catalog number: 21-031-CV )
  15. Sterile 0.25% trypsin/EDTA (Mediatech, catalog number: 22-053-CI )
  16. 4% buffered formalin solution (Sigma-Aldrich, catalog number: HT5012 )
  17. Prolong Gold anti-fade reagent plus DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36934 )
  18. Sterile Minimum Essential Medium without glutamine (Mediatech, catalog number: 15-010-CV )
  19. Sterile L-glutamine 100x (Mediatech, catalog number: 25-005-CI )
  20. Penicillin/Streptomycin 100x (Mediatech, catalog number: 30-002-CI )
  21. Fetal bovine serum (FBS) (Omega Scientific, catalog number: FB-11 )
  22. Doxycycline monohydrate (tetracycline analog) (Sigma-Aldrich, catalog number: D1822 )
  23. HEPES (Fisher Scientific, catalog number: BP310 )
  24. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  25. Anti-c-Myc monoclonal antibody, clone 9E10
    Note: Hybridoma cells from The Developmental Studies Hybridoma Bank (http://dshb.biology.uiowa.edu). Prepare mouse ascites using a commercial vendor.
  26. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A5666 )
    Note: This product has been discontinued.
  27. L-glycine (Fisher Scientific, catalog number: BP381-5 )
  28. Normal Donkey serum (LAMPIRE Biological Labs, catalog number: 7332100 )
  29. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  30. Mannose-6-Phosphate Receptor antibody (kind gift from William Brown, Cornell University)
  31. Fish skin gelatin (Sigma-Aldrich, catalog number: G7765 )
  32. Donkey anti-mouse DyLight 488 (Jackson ImmunoResearch Laboratories, catalog number: 715-485-150 )
    Note: This product has been discontinued.
  33. Donkey anti-rabbit DyLight 594 (Jackson ImmunoResearch Laboratories, catalog number: 711-515-152 )
    Note: This product has been discontinued.
  34. Complete media (see Recipes)
  35. Serum-free media (see Recipes)
  36. 1 mg/ml doxycycline stock solution (see Recipes)
  37. MEM ETC (see Recipes)
  38. MEM ETC containing anti-c-Myc antibody (see Recipes)
  39. Quench solution (see Recipes)
  40. Block and Permeabilization buffer (see Recipes)
  41. Primary antibody solution (see Recipes)
  42. Washing solution (see Recipes)
  43. Secondary antibody solution (see Recipes)

Equipment

  1. BSL-2 hood (Nuaire, class II)
  2. Tissue culture incubator at 37 °C and 5% CO2 (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150 )
  3. Orbital shaker (Bellco)
  4. Hemocytometer (Hausser Scientific, catalog number: 3200 )
  5. Surgical scissors 140 mm (Dunrite Instruments, catalog number: 140940 )
  6. Dissecting forceps, fine tip, non-serrated
  7. Olympus Fluoview FV1000S (Olympus, model: Fluoview FV1000 ) Spectral Laser Scanning Confocal microscope using an Olympus UPLFLN 60x oil-immersion objective
  8. Cold room (4 °C)

Software

  1. Adobe Photoshop (Adobe Systems Inc.)

Procedure

Notes:

  1. Perform Procedures A and B using aseptic technique in a BSL-2 biosafety cabinet.
  2. For Transwells, the upper chamber volume is 0.5 ml; the lower chamber volume is 1 ml.
  3. The volume of any solution added to membrane cut-out is 50 µl.

  1. Seed cells
    1. Starting with a 10 cm dish of MDCK cells in culture, aspirate complete media (see Recipes) and add 10 ml of DPBS without calcium or magnesium.
    2. Aspirate DPBS and add 1 ml of 0.25% trypsin/EDTA, place in a 37 °C incubator with 5% CO2 for 5 min occasionally tapping dish to dislodge cells.
    3. Add 9 ml of complete medium to dislodged cells in plate. Using a 10 ml serological pipette, pipette the cell suspension multiple times to detach the cells from each other.
    4. Use a hemocytometer to count the single-cell cell suspension.
    5. Dilute the cell suspension to a concentration of 50,000 cells/ml in complete media.
    6. Add 0.5 ml of cell dilution to the upper chamber of Transwell.
    7. Add 1 ml of complete media to the lower chamber of Transwell.
    8. Next day, change media to serum-free media (see Recipes) in the upper chamber. Keep complete media (serum-replete) in the lower chamber.
    9. After four days, add serum-free media containing doxycycline to the upper chamber and add complete media containing doxycycline to the lower chamber (dilute doxycycline stock solution [see Recipes] 1:20,000 to 50 ng/ml); incubate overnight.

  2. Antibody feeding
    1. Replace media on Transwells with ice-cold MEM ETC (see Recipes) and keep on ice.
    2. Add ice-cold MEM ETC containing anti-c-Myc antibody (see Recipes) to wells of a new 12-well plate and place on ice.
    3. Transfer each Transwell insert containing your cells to the new 12-well plate containing antibody on ice.
    4. Incubate on ice in the cold room for 20 min with gentle shaking.
    5. Remove plate from the cold room and aspirate antibody media from the lower chamber.
    6. Wash lower chambers with ice-cold MEM ETC three times for 5 min on ice in cold room with gentle shaking.
    7. Transfer each Transwell insert to a new 12-well plate containing pre-warmed MEM ETC in each well. Replace apical media with pre-warmed MEM ETC. Move to a 37 °C incubator.
    8. Incubate wells for the desired time, remove from the incubator, place in a new 12-well plate containing ice-cold MEM ETC, and store on ice until all time points are completed.
    9. Wash one time with ice-cold MEM ETC for 5 min on ice in cold room with gentle shaking. Remove media.
    10. Add 4% buffered formalin solution to upper and lower chambers; incubate at room temperature for 15 min with gentle shaking.
    11. Aspirate formalin and add quench solution (see Recipes) to both chambers and incubate at room temperature for 10 min with gentle shaking.
    12. Aspirate quench solution and wash both chambers with PBS three times.
    13. Add block and permeabilization buffer (see Recipes) to both chambers and incubate at 37 °C for 30 min.

  3. Primary antibody (M6PR antibody) incubation
    1. Aspirate blocking buffer, remove Transwell inserts from the plate, and place upside-down (apical side facing downward) on bench top.
    2. Use a new razor blade to cut three sides of a rectangle in the Transwell membrane.
    3. Gently grab the square membrane with forceps and cut the rectangle from the Transwell support using surgical scissors. Clip a small piece from the upper right corner to be able to keep track of which side the cells are on. (Video 1)

      Video 1. Demonstration of the procedure to cut out the Transwell membrane using a razor blade. The lid of the multiwall dish is covered with Parafilm and used as a support surface.

    4. Place the membrane cut-out, cell side up, onto a sheet of Parafilm placed in a lid of a 12-well plate with each well labeled.
    5. Gently add 50 µl of primary antibody solution (see Recipes) on top of membrane cut-out.
    6. Place samples in a plastic pencil box lined with damp paper towels to create a humid chamber and incubate overnight in the cold room.

  4. Secondary antibody incubation
    1. Aspirate primary antibody solution, transfer membranes to a new 12-well plate, and wash four times each for 5 min with washing solution (see Recipes).
    2. Add secondary antibody solution (see Recipes) to membranes, incubate at 37 °C in the humid chamber for 1 h.
    3. Aspirate secondary antibody solution and wash three times 5 min with washing solution.
    4. Wash once with PBS.
    5. Aspirate PBS, and post-fix by adding 4% buffered formalin solution, and incubate at room temperature for 10 min.
    6. Aspirate formalin, wash one time with PBS quickly, and another time for 5 min.
    7. Aspirate PBS, use forceps to dip membrane in ddH2O for 1 sec. Remove from ddH2O, wick water away by gently touching membrane to a Kimwipe, and place on microscope slide cell-side up. Keep membrane flat the entire time preventing it from folding back on itself.
    8. Add a drop of mounting medium (Prolong Gold anti-fade reagent plus DAPI) to each membrane.
    9. Gently add rectangular coverslip to membranes avoiding air bubbles.
    10. Store slides in the dark at room temperature for 48 h to harden mounting media.

Data analysis

  1. Analyze using a confocal microscope.
  2. Acquire high-quality images of representative fields from focal planes corresponding to different areas of the cell mono-layer (nuclear level, apical cytoplasm, apical plasma membrane).
  3. Acquire corresponding images from each time point.
  4. Assemble micrographs in Adobe Photoshop.
  5. Results in Figure 2.


    Figure 2. Example of immunofluorescence microscopy of a basolateral antibody feeding experiment. MDCK cells stably transfected for wild-type Stx3 were grown at to post-confluence for 4 days on Transwell filters, and then induced with DOX for 16 h. Anti-myc antibody (9E10 ascites) was added to the basolateral media and incubated at 4 °C before being washed off and incubated at 37 °C for 5 min. Cells were fixed and stained for Stx3 (myc), green; M6PR, red; and nuclei (DAPI), blue. Scale bar = 10 μm.

Notes

To perform the apical antibody-feeding experiment, repeat the protocol but add MEM ETC containing anti-c-Myc ascites to the upper chamber instead of the lower chamber.

Recipes

  1. Complete media (500 ml)
    To a new bottle of Minimum Essential Media, add:
    5 ml L-glutamine
    5 ml penicillin/streptomycin
    50 ml of FBS
  2. Serum-free media (500 ml)
    To a new bottle of Minimum Essential Media, add:
    5 ml L-glutamine
    5 ml penicillin/streptomycin
  3. 1 mg/ml doxycycline stock solution (store at -20 °C)
    Add 50 mg of doxycycline to 50 ml of ddH2O
  4. MEM ETC (Minimum Essential Medium + 20 mM HEPES + 0.6% BSA + Pen/Strep)
    1. To Minimum Essential Medium with penicillin/streptomycin add 1 M HEPES-NaOH (pH 7.4) to a final concentration of 20 mM
    2. Add BSA to a final concentration of 0.6%
  5. MEM ETC containing anti-c-Myc antibody ascites (1:200)
    Add 50 µl of mouse ascites with 9E10 anti-c-Myc antibody to 10 ml of MEM ETC
  6. Quench solution (75 mM NH4Cl + 20 mM L-glycine)
    200.6 mg of NH4Cl
    75.1 mg L-glycine
    Bring to volume 50 ml with PBS
  7. Block and Permeabilization buffer (PBS containing 0.2% Triton X-100, 5% Normal Donkey serum)
    1 ml of Normal Donkey serum
    2 ml of 10x PBS
    0.4 ml of 10% Triton X-100 stock
    Bring to volume 20 ml with ddH2O
  8. Primary antibody solution
    Add 3 µl of anti-M6PR (1:200) to 600 µl of Block and Permeabilization buffer
  9. Washing solution (PBS containing 0.05% Triton X-100, 0.7% fish skin gelatin)
    70 mg of fish skin gelatin
    50 µl of 10% Triton X-100
    1 ml of 10x PBS
    Bring to volume 10 ml with ddH2O
  10. Secondary antibody solution
    Add 1 µl of donkey anti-mouse DyLight 488 (1:1,000) and 1 µl of donkey anti-rabbit DyLight 594 (1:1,000) to 1 ml of Block and Permeabilization buffer

Acknowledgments

This work was supported by grants from the NIH (DK62338), and the California Cancer Research Coordinating Committee to T.W., by a grant BFU2012-35067 from the Spanish Ministry of Economy and Competitiveness (MINECO) to A.F.-R., and a Postdoctoral Fellowship from the Spanish Ministry of Education and Science to E.R. The authors declare no conflicts of interest or competing interests.

References

  1. Delgrossi, M. H., Breuza, L., Mirre, C., Chavrier, P. and Le Bivic, A. (1997). Human syntaxin 3 is localized apically in human intestinal cells. J Cell Sci 110 (Pt 18): 2207-2214.
  2. Giovannone, A. J., Reales, E., Bhattaram, P., Fraile-Ramos, A. and Weimbs, T. (2017). Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes. Mol Biol Cell 28(21): 2843-2853.
  3. Kreitzer, G., Schmoranzer, J., Low, S. H., Li, X., Gan, Y., Weimbs, T., Simon, S. M. and Rodriguez-Boulan, E. (2003). Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells. Nat Cell Biol 5(2): 126-136.
  4. Li, X., Low, S. H., Miura, M. and Weimbs, T. (2002). SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes. Am J Physiol Renal Physiol 283(5): F1111-1122.
  5. Low, S. H., Chapin, S. J., Weimbs, T., Komuves, L. G., Bennett, M. K. and Mostov, K. E. (1996). Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. Mol Biol Cell 7(12): 2007-2018.
  6. Low, S. H., Chapin, S. J., Wimmer, C., Whiteheart, S. W., Komuves, L. G., Mostov, K. E. and Weimbs, T. (1998). The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells. J Cell Biol 141(7): 1503-1513.
  7. Low, S. H., Miura, M., Roche, P. A., Valdez, A. C., Mostov, K. E. and Weimbs, T. (2000). Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity. Mol Biol Cell 11(9): 3045-3060.
  8. Low, S. H., Vasanji, A., Nanduri, J., He, M., Sharma, N., Koo, M., Drazba, J. and Weimbs, T. (2006). Syntaxins 3 and 4 are concentrated in separate clusters on the plasma membrane before the establishment of cell polarity. Mol Biol Cell 17(2): 977-989.
  9. Reales, E., Sharma, N., Low, S. H., Folsch, H. and Weimbs, T. (2011). Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity. PLoS One 6(6): e21181.
  10. Sharma, N., Low, S. H., Misra, S., Pallavi, B. and Weimbs, T. (2006). Apical targeting of syntaxin 3 is essential for epithelial cell polarity. J Cell Biol 173(6): 937-948.
  11. ter Beest, M. B., Chapin, S. J., Avrahami, D. and Mostov, K. E. (2005). The role of syntaxins in the specificity of vesicle targeting in polarized epithelial cells. Mol Biol Cell 16(12): 5784-5792.
  12. Weimbs, T., Low, S. H., Chapin, S. J. and Mostov, K. E. (1997). Apical targeting in polarized epithelial cells: There's more afloat than rafts. Trends Cell Biol 7(10): 393-399.

简介

细胞表面受体的摄取和贩卖可以通过被称为“抗体 - 进纸”技术,其在外部使用上应用的抗体来标记培养的活细胞的表面上的受体进行监测。在这里,我们适应传统的抗体喂养实验极化上皮细胞(Madin-Darby犬肾)生长在透性transwell支持。通过添加两个串联胞外Myc表位标签的SNARE蛋白突触3(Stx3)的C末端,我们提供了一种站点,其中与抗体可以结合,使我们能够用不同的顶端和基底外侧膜对细胞进行抗体 - 喂养实验。通过这个程序,我们观察到Stx3的内吞和细胞内运输。具体而言,我们评估从基底外侧膜Stx3的内在化速率和在时间和空间上使用固定在不同时间点的细胞免疫荧光显微镜随后的内吞观察路径。为此,介绍了以下可追踪单层的贩运行为:来自限制膜的靶蛋白。

【背景】SNARE蛋白突触3(Stx3)是已知的建立在极化上皮细胞顶端 - 基底极性(低等人,1996年Delgrossi 等人,1997年Weimbs 等人,1997;低等人,1998;黎等人,2002;低等人
“”
一个典型的12孔细胞培养皿的阱内的transwell小室的聚碳酸酯膜的细胞培养插入物的图1示意图。斯特朗细胞是在膜的顶部和培养将形成紧密的单层做密封关聚碳酸酯膜将顶端介质隔室与基底外侧介质隔室分开。

关键字:抗体喂养试验, 突触融合蛋白, SNARE, 内化, 顶端 - 基底外侧极性, 上皮细胞, MDCK细胞

材料和试剂

  1. 过滤器尖端,透明,无菌F. Gilson P1000,60盒/箱(Greiner Bio One International,目录号:740288)
  2. Gilson P-200,96 PCS /盒(Greiner Bio One International,目录号:739288)。
  3. 标准过滤器尖端,20微升,透明,通用,无菌,每个机架96件(Greiner Bio One International,目录号:774288)。
  4. 土壤研磨SK38 2毫升管(Bertin Technologies,目录号:KT03961-1-006.2)。
  5. 的Eppendorf ®管3810X,将1.5ml,G-安全®离心稳定性,Eppendorf优质 TM ,无色,1000个。 (Eppendorf,目录号:0030125150)
  6. 无水乙醇,EMSURE ACS,ISO,Reag。 Ph.Eur。分析试剂(Merck,Millipore Sigma,目录号:1.00983.1000)
  7. UltraPure TM DNase / RNase-Free蒸馏水(Thermo Fisher Scientific,Gibco TM,目录号:10977035)。
  8. QIAamp Fast DNA Stool Mini试剂盒(QIAGEN,目录号:51604),包含以下内容:
    1. QIAamp迷你旋转柱
    2. 收集管(2毫升)
    3. InhibitEX Buffer
    4. 蛋白酶K
    5. 缓冲区AL
    6. 缓冲AW1集中
    7. 缓冲AW2浓缩液
    8. 缓冲区ATE

设备

  1. Finnpipette F1,100至1,000μl(Thermo Fisher Scientific,目录号:4641100N)。
  2. Finnpipette F1,20至200μl(Thermo Fisher Scientific,目录号:4641080N)
  3. Finnpipette F1,2至20μl(Thermo Fisher Scientific,目录号:4641060N)。
  4. 烧杯
  5. -80°C冷冻机
  6. Precellys 24(Bertin Technologies,目录号:EQ03119.200.RD00.0)
  7. 带有加热块的Thermoshaker用于24 x1.5 ml微管(Grant Instruments,目录号:PHMT-PSC24N)
  8. 具有用于1.5 / 2ml管的转子的微量离心机5417R(Eppendorf,型号:5417R,目录号:2262 180-7)。
  9. 涡旋混合器(Merck Eurolab,目录号:MELB 1719)
  10. NanoDrop分光光度计

程序

  1. 粪便样品收集
    1. 从小鼠中收集1-2个新鲜的粪便颗粒到土壤研磨的SK38 2ml试管中。一只手握住老鼠收集新鲜的粪便颗粒,在此过程中,可以直接将粪便排入另一只手握住的SK38 2ml试管中。或者,可将个别小鼠放入无菌烧杯中几分钟,然后从烧杯中收集新鲜的粪粒。重要的是要在一天中在Sametime周期实验队列内收集从所有动物粪便样品是重要的,因为它其中报告DASS模具以及微生物群组成显示昼夜振荡(Thaiss 等人,2014)。
    2. 将含有粪便颗粒的土壤研磨SK38 2ml试管储存在-80℃直至进一步处理。粪便DNA提取,因此可以从新鲜的粪便颗粒立即开始,但内实验队列所有样品应该是所有新鲜或全部冻结,以避免存储的工件,如冷冻其中显示影响厚壁菌门的<比率/粪便样品 to Bacteroidetes (Bahl et。,2012)。

  2. 粪便DNA提取
    粪便DNA提取用QIAamp 执行的®快速DNA粪便Mini试剂盒gemäß制造商的与由珠磨额外的机械裂解指令详述如下:
    1. 加入1ml InhibitEX缓冲液到含有粪便颗粒的土壤研磨SK382ml管中。取自-80°C冰柜的粪便颗粒可以立即使用,因为不需要。
    2. 均质粪便颗粒在1ml Inhibitex ®缓冲器通过使用Precellys珠磨® 24在6500转2×30秒。
    3. 将步骤B2的粪便匀浆转移到无菌的1.5ml微量离心管中,注意避免转移锆珠。
    4. 在70℃加热5分钟的热匀浆中的粪便匀浆,同时在1100rpm振荡,以促进细菌的进一步裂解。
    5. 将来自步骤B4的粪便匀浆在室温下在20,800×gg下离心1分钟以沉淀粪便颗粒。
    6. 在平均时间,移液管15微升蛋白酶K从QIAamp试剂®快速DNA粪便Mini试剂盒到新鲜无菌1.5ml微量管中。
    7. 吸取步骤B5的200μL粪便匀浆上清液到含有1.5ml微量离心管的蛋白酶K中。
    8. 添加200μl缓冲液AL到1.5ml微量管中含有200微升上清液,蛋白酶K(来自步骤B7),并涡旋15秒,以形成均匀的悬浮液。

    9. 在70℃加热10分钟,同时摇动1,100转

    10. 在悬浮液中加入200μl无水乙醇并涡旋混合
    11. QIAamp Fast DNA Stool Mini Kit中提供的QIAamp离心柱。
    12. 关闭QIAamp离心柱,并在室温下以20,800×g g离心1分钟。
    13. 将QIAamp离心柱置于新的2 ml收集管中,丢弃含有滤液的旧收集管。
    14. 向QIAamp离心柱中加入500μl缓冲液AW1。
      注意:缓冲区AW1是作为浓缩物提供的。当使用新鲜的瓶子时,首先将25毫升无水乙醇加入到AW1浓缩物中,并通过重悬来彻底混合。
    15. 关闭QIAamp离心柱,并在20,800×gg室温下离心1分钟。
    16. 将QIAamp离心柱置于新的2 ml收集管中,丢弃含有滤液的旧收集管。
    17. 向QIAamp离心柱中加入500μl缓冲液AW2。
      注意:Buffer AW2是作为浓缩物提供的。当使用新鲜的瓶子时,首先将30毫升无水乙醇加入到AW2浓缩物中,并通过重悬来彻底混合。
    18. 关闭QIAamp离心柱,并在室温下以20,800×g g离心3分钟。
    19. 将QIAamp离心柱置于新的2 ml收集管中,丢弃含有滤液的旧收集管。
    20. 为了减少缓冲AW2残留的机会,在室温下以20,800xg克再次离心3分钟。
    21. 将QIAamp离心柱置于新鲜的1.5ml微量离心管中,丢弃含有滤液的旧收集管。
    22. 向QIAamp离心柱中加入200μlUltraPure™DNase / RNase-Free蒸馏水以洗脱DNA。
      注:“DNA可以因此使用200微升的缓冲液洗脱ATE通过QIAamp试剂 ® 快速DNA粪便Mini试剂盒提供。 /

    23. 在室温下孵育1分钟

    24. 在20,800×g的室温下离心1分钟以收集DNA。
    25. 丢弃QIAamp旋转栏。
    26. 关闭含有洗出液的1.5ml微量离心管,并将DNA储存在-20℃直至进一步使用。这个协议获得的粪便DNA的数量因样本而异,并且取决于用于提取DNA的粪便的量。在我们的手中,获得的粪便DNA的量在每毫升粪便50-500ng DNA之间变化,平均每mg粪便约150ng DNA。由于一个粪便颗粒重量至少为20毫克,所以使用这个方案可以获得至少1微克的粪便DNA。因此,当粪便DNA在200μl的超纯水 TM DNA酶/ RNA酶的蒸馏水(见步骤A22),粪便DNA的从任何给定的样品所获得的浓度将是至少5纳克的溶解/μl。

数据分析

将所得到的粪便DNA的浓度和质量可以通过纳米滴分析来测定,其中,在260nm / 230 nm和260nm处/ 280nm的吸光度比可以是确定性的开采评价提取的DNA的纯度,这应该是围绕两个和1.8,分别。下游系统发育16S rDNA微生物群分析始于对25ng粪便DNA的PCR。因此,由于该方案以5ng /μl的浓度产生至少1μg的DNA,所获得的DNA量不是限制因素。进一步的系统发育16S rDNA微生物群分析程序在我们以前的研究中概述(Mamantopoulos等,2017)。

确认

A. W.通过从科学研究基金龙龙的奥德修斯授予G.0C49.13N支持,是一个博士后与科研龙龙基金。作者没有任何利益冲突。

参考

  1. Bahl,M.I.,Bergstrom,A。和Licht,T.R。(2012)。 之前DNA提取冻结的粪便样品的影响的厚壁菌至 。拟杆菌比确定性由下游定量PCR分析代码开采 FEMS微生物学快报 329(2):193-197
  2. 科斯泰亚,PI,泽勒,G.砂川,S.,佩尔蒂埃,E.,阿尔贝蒂,A.,Levenez,F.,特拉蒙塔诺,M.,Driessen的,M.,Hercog,R.,杨,FE,Kultima, JR,海沃德,MR,科埃略,LP,艾伦 - Vercoe,E.贝特朗,L.,Blaut,M.,棕色,JRM,纸箱,T.,库尔斯看门,S.,Daigneault,M.,Derrien, M.,Druesne,A.,德沃思,WM,芬雷,BB,火石,HJ,瓜内尔,F.,服部,M.,圣,H.,露娜,RA,面包车Hylckama Vlieg,J.,Junick,J ,Klymiuk,一,兰杰拉,P.,勒夏特列,E.,五月五,Manichanh,C.,马丁,JC,梅里,C.,森田,H.,奥图尔,PW,Orvain, C.,帕蒂尔,KR,Penders的,J.,Persson的,S.脑桥,N.,波波娃,M.,萨洛宁,A.,Saulnier,D.,斯科特,KP,辛格,B.,斯莱扎克,K. ,Veiga的,P.,Versalovic,J.,召,L.,Zoetendal,EG,埃利希,SD,多尔,J。和博克,P。(2017)。 走向在宏基因组研究人类粪便样品的处理标准。 纳特生物技术 35(11):1069-1076。
  3. Ferrand的,J.,赞助,K.,罗格朗Frossi,C.,Frippiat,J.P.,梅林,C.,Alauzet,C。和Lozniewski,A。(2014)。的七种方法从小鼠的粪便和盲肠样品细菌DNA提取比较。 J Microbiol Methods 105:180-185。
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  5. 萨洛宁,A.,Nikkila,J.,Jalanka-Tuovinen,J. Immonen,O.,Rajilic-Stojanovic的,M.,吉科宁,R.A.,帕尔瓦(Palva),A。和德沃思,W. M.(2010)。使用机械细胞细菌和古细菌的DNA的有效回收:粪便DNA提取方法与系统发育的微阵列比较分析。裂解代码Ĵ微生物学方法 81(2):127-134
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引用:Giovannone, A. J., Reales, E., Bhattaram, P., Fraile-Ramos, A. and Weimbs, T. (2018). Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells. Bio-protocol 8(3): e2453. DOI: 10.21769/BioProtoc.2453.
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