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Isolation and Quantification of Plant Extracellular Vesicles
植物细胞外囊泡的分离和定量   

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Plant Physiology
Jan 2017

Abstract

Extracellular vesicles (EVs) play an important role in intercellular communication by transporting proteins and RNA. While plant cells secrete EVs, they have only recently been isolated and questions regarding their biogenesis, release, uptake and function remain unanswered. Here, we present a detailed protocol for isolating EVs from the apoplastic wash of Arabidopsis thaliana leaves. The isolated EVs can be quantified using a fluorometric dye to assess total membrane content.

Keywords: Arabidopsis thaliana (拟南芥), Extracellular vesicles (细胞外囊泡), EVs (Evs), Apoplastic wash (质外体洗涤), DiOC6 (DiOC6), Fluorometric quantification (荧光定量)

Background

Extracellular vesicles (EVs) are membrane-bound structures that mediate the cell-to-cell transfer of proteins, lipids and genetic material. Interest in mammalian EVs has grown over the years due to their ability to transfer RNA and modulate immune responses. Mammalian EVs are routinely isolated for study from the medium of cultured cells, as well as a growing list of biological fluids (Colombo et al., 2014). Plant EVs are also thought to have a role in the immune response but are comparatively understudied (An et al., 2007; Davis et al., 2016). This is due, in large part, to the absence of a method of isolation.

While plant EVs have been observed since 1967 using transmission electron microscopy, methods for their isolation were not developed until 2009 (Halperin and Jensen, 1967). Regente et al. (2009) isolated small (50-200 nm in diameter) vesicle-like structures from water-imbibed sunflower (Helianthus annuus) seeds. We modified the methods presented in Regente et al. (2009) to isolate vesicles from the apoplastic wash of Arabidopsis thaliana rosettes. To determine which conditions induce or impair EV secretion, we also designed a method for staining the EV pellet with 3,3’-dihexyloxacarbocyanine iodide (DIOC6(3)), a fluorescent lipophilic dye. In the absence of sophisticated forms of nanoparticle tracking, this relatively simple approach quantifies the total membrane content and can be used to indirectly measure the concentration of EVs (Rutter and Innes, 2017). For more precise measurements, and to assess the size distributions of EVs, nanoparticle tracking can be used. Our protocols enable the study of plant EV content and composition, as well as the pathways and conditions that mediate EV biogenesis and release.

Materials and Reagents

  1. MicroporeTM surgical tape (3M, catalog number: 1530-1 )
  2. Clear plastic domes (Hummert International, catalog number: 11-3348 )
  3. 30 ml needle-less syringes (BD, catalog number: 309650 )
  4. 50 ml conical tubes (VWR, catalog number: 89039-656 )
  5. Pipette tips
  6. Paper towels
  7. 250 ml plastic bottles (w/out cap) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3120-0250 )
  8. 15 ml conical tube (VWR, catalog number: 89039-668 )
  9. 1.5 ml microcentrifuge tubes (VWR, catalog number: 20170-038 )
  10. 10 ml syringe
  11. 5 ml needle-less syringes (BD, catalog number: 309646 )
  12. Acrodisc 0.45 μm syringe filter (Pall, catalog number: 4454 )
    Note: An Acrodisc 0.22 μm filter (Pall, catalog number: 4192 ) can also be used if one is concerned about bacterial contamination.
  13. Pryme PCR 8 strip 0.2 ml tubes (MIDSCI, catalog number: AVSST )
  14. Kim-wipes (KCWW, Kimberly-Clark, catalog number: 34120 )
  15. COSTAR EIA/RIA Plate, 96 well, half area, no lid, flat bottom, non-treated, black polystyrene plate (Corning, Costar®, catalog number: 3694 )
  16. 5.8 ml transfer pipets (Thermo Fisher Scientific, catalog number: 222-1S )
  17. Petri dishes, 100 x 15 mm (VWR, catalog number: 25384-302 )
  18. Arabidopsis seeds
  19. Bleach (Austin’s, catalog number: 90000360 )
  20. PRO-MIX PGX Biofungicide potting mix (Premier Tech Horticulture, catalog number: 10382RG )
  21. OptiprepTM density gradient medium (Sigma-Aldrich, catalog number: D1556 )
  22. Murashige Skoog basal salt mixture (Sigma-Aldrich, catalog number: M5524 )
  23. Sodium hydroxide (NaOH) pellets (Avantor Performance Materials, MACRON, catalog number: 7708-10 )
  24. Potassium hydroxide (KOH) pellets (Fisher Scientific, catalog number: P250-500 )
  25. Agar (Sigma-Aldrich, catalog number: 05040 )
  26. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
  27. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  28. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  29. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  30. Hydrochloric acid (HCl) (EMD Millipore, catalog number: HX0603-3 )
  31. 3,3’-Dihexyloxacarbocyanine iodide (DiOC6(3)) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D273 )
  32. Plant protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
  33. 2,2’-Dipyridyl disulfide (DPDS) (Sigma-Aldrich, catalog number: 149225 )
  34. 0.5x Murashige and Skoog agar (see Recipes)
  35. Vesicle isolation buffer (VIB) (see Recipes)
  36. 20 mM Tris-HCl, pH 7.5 (see Recipes)
  37. DiOC6 staining solution (see Recipes)
  38. Vesicle resuspension buffer (see Recipes)

Equipment

  1. Metal forceps
  2. Scissors
  3. Stainless steel tweezers, type 3 (Techni-Tool, catalog number: 758TW474 )
  4. Scale
  5. 1 L plastic beakers
  6. Titanium French press coffee maker, 24 fl oz (Snow Peak, catalog number: CS-111 )
  7. Vacuum chamber, 0.20 Cu. Ft. (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F42031-0000 )
  8. Vacuum pump, 3.5 CFM (Ideal Vacuum Products, catalog number: P101532 )
  9. JA-14 fixed-angle rotor (Beckman Coulter, model: JA-14, catalog number: 339247 )
  10. Pipettes
  11. MJ research PTC-200 thermal cycler (MJ Research, catalog number: 8252-30-0001 )
  12. Fluorometric microplate reader
    Note: We use an AppliskanTM fluorometric plate reader (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5230000 ) to measure DiOC6 fluorescence, but any fluorometer capable of detecting multiple wavelengths could work.
  13. SW 41 Ti rotor, swinging bucket, titanium, 6 x 13.2 ml, 41,000 rpm, 288,000 x g (Beckman Coulter, model: SW 41 Ti, catalog number: 331362 )
  14. Thickwall polycarbonate centrifuge tubes (3.5 ml, 13 x 51 mm) (Beckman Coulter, catalog number: 349622 )
  15. Thinwall, Ultra-ClearTM 13.2 ml, 14 x 89 mm ultracentrifuge tubes (Beckman Coulter, catalog number: 344059 )
  16. TLA100.3 fixed-angle rotor (Beckman Coulter, model: TLA-100.3, catalog number: 349481 )
  17. Avanti J-26S XP centrifuge (Beckman Coulter, model: Avanti J-26S XPI , catalog number: B22989)
  18. Fluorescent light microscope
  19. Growth chamber
  20. Ice bucket (schuett-biotech, Spongex, catalog number: 3.680 052 )
  21. Optima TLX ultracentrifuge (Beckman Coulter, model: OptimaTM TLX , catalog number: 361545)
  22. Nanoparticle trackers (Particle Metrix, model: ZetaView® , or Malvern Instruments, model: Nanosight NS300 )

Procedure

  1. Preparing the plants
    1. Sterilize Arabidopsis seeds with 50% Bleach for no more than 5 min and wash three times with sterile dH2O. Each wash step should last 1-2 min.
    2. Plate the seeds on 0.5x Murashige and Skoog (MS) medium containing 0.8% agar (see Recipes).
    3. Wrap medical tape around the circumference of each MS plate and store the plates in the dark at 4 °C for 2 days.
    4. After 2 days, move the plates to short day conditions (9-h days, 22 °C, 150 μEm-2). Store the plates vertically and allow the seeds to germinate and grow for 7 days.
    5. Transfer the seedlings to PRO-MIX PGX. Cover the seedlings with a clear plastic dome for one week. On day 7, crack the dome. The next day, remove the dome completely. Water twice a week or as needed. Allow the plants to grow for 6 weeks total before harvest.
    Note: Growing seedlings on MS plates first and transferring them to soil later ensures an exact number of healthy plants for each experiment. A typical experiment requires ~36 plants for each genotype/treatment. We have found that six-week-old plants yield the optimal amount of intercellular wash fluid with the smallest risk of cellular contamination.

  2. Preparing tools for apoplastic wash collection
    1. Using metal forceps heated in a flame, melt 8 additional holes at the end of a 30 ml syringe (Figure 1 and Video 1).
      Note: These additional channels allow for the optimal collection of apoplastic wash and provide an alternate route should the primary hole become plugged.
    2. Use the heated forceps to perforate the cap of a 50 ml conical tube multiple times, creating a circle ~2.5 cm in diameter. Punch out the circle to create a hole in the middle of the cap. Soften the cap in a flame and slide it onto the 30 ml syringe until it is ~1.5-2 cm from the top. The syringe can now be screwed securely onto a 50 ml conical tube (Figure 1 and Video 1).
      Note: This prevents the end of the syringe from becoming submerged in the apoplastic wash.


      Figure 1. Tool for apoplastic wash collection. Apoplastic wash is collected by packaging whole Arabidopsis thaliana rosettes in a 30 ml syringe. Eight additional holes are melted into the end of the syringe with forceps to create channels for the wash to flow through. A 2.5 cm hole is punched out of a 50 ml conical tube cap. The cap is then slid onto the syringe 2 cm from the top. This allows the syringe to be screwed securely into a 50 ml conical tube. See Video 1.

      Video 1. EV isolation demonstration. This video demonstrates how to prepare the syringe and conical tube apparatus shown in Figure 3, and the procedures for extracting apoplastic wash fluid from leaves and subsequent purification of EVs from this fluid.

  3. Collecting the apoplastic wash
    Note: An overview of the workflow from apoplastic wash collection to DiOC6 quantification can be seen in Figure 2.


    Figure 2. General workflow. Apoplastic wash is collected by vacuum infiltrating full Arabidopsis rosettes with an isotonic buffer, washing out the infiltrated buffer using a low-speed centrifugation spin and filtering the wash to remove any large debris. The wash fluid is then processed using differential centrifugation to isolate EVs, which can be stained with the lipophilic dye DiOC6. After the EVs are washed and pelleted again to remove excess dye, DiOC6 fluorescence in the EV pellet can be measured to indirectly quantify the amount of EVs.

    1. Using scissors cut each Arabidopsis plant off at the root.
      Note: Leaving a little of the root will help later on with manipulating the rosettes. See Video 1.
    2. Collect the harvested rosettes in a 1 L plastic beaker and record the mass.
      Note: Include three biological replicates for each treatment/genotype. The replicates should all be the same volume and should not be less than 0.5 ml. It takes ~3 g of rosettes to collect ~0.5 ml of apoplastic wash.
    3. Rinse the rosettes 3 times with water to remove soil particles.
    4. Carefully place the rosettes in the French press with 300-500 ml of vesicle isolation buffer (VIB) (see Recipes). Place the lid on the French press and gently lower the plunger until the plants are fully submerged.
    5. Place the French press, plants and buffer in a vacuum chamber. Apply the vacuum for 20 sec.
    6. Remove the French press from the vacuum chamber, take the lid off and pour the VIB into a separate 1 L plastic beaker. Remove the rosettes from the French press.
      Note: The VIB can be reused multiple times on the same day, but, once used for vacuum infiltration, the VIB should not be stored for use on another day.
    7. Remove excess VIB from the plants by gently shaking them by the roots and then brushing the leaves across a paper towel.
    8. Place the rosettes root down on top of the opening of a 30 ml syringe (Figure 3A). Gently prod and coax the rosettes into the syringe without crushing or tearing the leaves (Figure 3B). Once the rosettes are in far enough, tapping the syringe on a hard surface will force them fully into the syringe (Figure 3C).
      Note: The rosettes can be inserted in stacks of 2 or 3. Typically, 4-6 six-week-old rosettes will fit inside a 30 ml syringe. The rosettes should not be so tightly packed as to damage the tissues or obstruct the flow of apoplastic wash out of the syringe. See Video 1.
    9. Screw the syringe into a 50 ml conical tube (Figure 3D) and place the tube and syringe in a 250 ml plastic bottle (Figure 3E). The cap secured around the top of the syringe should prevent the tube from falling into the bottle.


      Figure 3. Packaging rosettes for apoplastic wash extraction. A. The rosettes are placed root-down on the top of the syringe. B. The rosettes are gently prodded into the syringe. C. The syringe is tapped on a hard surface to force the rosettes into it. D. The syringe is screwed into a 50 ml conical tube. E. The rosettes, syringe and 50 ml conical tube is placed into a 250 ml plastic bottle, which is then placed into a JA-14 rotor for centrifugation. See Video 1.

    10. Place the syringe, 50 ml conical tube and bottle into the slot of a centrifuge rotor and centrifuge for 20 min at 700 x g, 4 °C.
      Note: This step is designed for a JA-14 fixed-angle rotor.
    11. Remove each syringe and conical tube from the centrifuge while carefully maintaining the tube’s angle. Unscrew the syringe and remove the apoplastic wash from the bottom of the tube. Make sure to avoid any pelleted soil particles. Transfer the apoplastic wash to a sterile 15 ml conical tube and keep the sample on ice.
    12. Using a 10 ml syringe, filter the apoplastic wash through a 0.45 μm filter into a new, sterile 15 ml conical tube.
      Note: The filtration step is a precaution meant to remove large pieces of debris. This step can result in the loss of apoplastic wash and can be skipped if the volume of apoplastic wash is small (~500 μl). In these cases, the 10,000 x g centrifugation should be enough to remove any debris.

  4. EV isolation and DiOC6 staining
    1. Transfer each volume of apoplastic wash to ultracentrifuge tubes. Bring the volume in each tube up to the tube’s maximum volume using chilled VIB.
    2. Centrifuge for 30 min at 10,000 x g, 4 °C.
    3. Carefully, transfer the supernatants to new ultracentrifuge tubes using a pipet.
      Note: Maintain the position of the tube after centrifugation and be careful to angle the pipet away from any debris that may have pelleted.
    4. Centrifuge for 60 min at 40,000 x g, 4 °C.
    5. Decant the supernatant and remove any excess buffer from the opening of the tube with a pipet. Resuspend the pellet in 100 μl of 100 μM DiOC6 in 20 mM Tris-HCl pH 7.5 (see Recipes). Add 1% plant protease inhibitor cocktail (PIC) and 0.2 mM DPDS to the DiOC6 staining solution (see Recipes) if the EVs will be used for an immunoblot after DiOC6 quantification.
      Note: When using small volumes of apoplastic wash, the EV pellet is invisible. A thin, translucent pellet can appear when using large volumes of apoplastic wash (≥ 12 ml). To effectively resuspend the pellet, use a pipet to repeatedly wash the side of the tube containing the pellet with half the added DiOC6 solution.
    6. Transfer the resuspended vesicles to a PCR tube and heat at 37 °C for 10 min in a thermal cycler.
    7. Transfer the sample to a new, clean ultracentrifuge tube and bring the volume up to the tube’s maximum volume using chilled 20 mM Tris-HCl pH 7.5.
    8. Centrifuge for 60 min at 40,000 x g, 4 °C.
    9. Decant the supernatant and use a pipet followed by a Kim-wipe to remove excess buffer for the opening and upper regions of the tube.
      Note: It is important to wipe out remaining buffer immediately after decanting, while the centrifuge tube is still upside down. Carefully removing all excess buffer ensures that each pellet is resuspended in the same total volume.
    10. Resuspend the pellet in 52 μl of vesicle resuspension buffer (20 mM Tris-HCl pH 7.5, see Recipes).
      Note: Add 1% plant protease inhibitor cocktail (PIC) and 0.2 mM DPDS to the vesicle resuspension buffer if the EVs will be used for an immunoblot after DiOC6 quantification.

  5. Collecting fluorometric data
    1. Add 50 μl of each sample to wells in a black polystyrene, 96-well, half-area, flat-bottom EIA/RIA plate.
      Note: For a typical experiment, there are three kinds of samples: a negative control of buffer (20 mM Tris-HCl + 1% plant PIC and 0.2 mM DPDS) to account for background signal, a positive control to determine vesicle secretion under mock conditions or in a genetically wild-type background, and the test sample to determine the effect of a treatment or mutation on vesicle secretion. As previously stated, include three biological replicates for each sample.
    2. Insert the plate into the fluorometric microplate reader. Excite the samples at 485 nm and record fluorescent emissions at 535 nm. Take three readings of each plate.
      Note: After recording fluorescence, the samples remain useful for a number of different assays, including staining for transmission electron microscopy, fluorescent light microscopy and immunoblots.

  6. EV purification
    Note: Differential centrifugation yields a crude EV preparation that may contain other components of the apoplast. In-depth studies of EV composition require a further purification. The following protocol is designed to separate EVs out from other contaminants using a discontinuous iodixanol density gradient. An overview of the purification process can be seen in Figure 4.


    Figure 4. Workflow for purification of EVs. Crude EVs are loaded on top of a discontinuous iodixanol gradient and centrifuged for 17 h at 100,000 x g, 4 °C. Following centrifugation, three EV-containing fractions are isolated. These fractions are washed and re-pelleted to remove the iodixanol. All three samples of EVs are then combined into a single tube, washed and pelleted to create one purified EV sample.

    1. Bring the volume of the isolated, crude EV preparation to 0.5 ml using sterile, cold VIB. Leave the sample on ice.
    2. Prepare iodixanol solutions of 40% (3 ml, v/v), 20% (3 ml, v/v), 10% (3 ml, v/v), and 5% (2 ml, v/v) using aqueous 60% iodixanol (OptiPrep) and sterile, cold VIB.
    3. Using a transfer pipet, add the iodixanol solutions to an ultracentrifuge tube. Carefully layer the solutions on top of one another, starting with the 60% solution and ending with the 5% solution. Lastly, add the 0.5 ml of crude EVs to the top of the gradient.
    4. Centrifuge in a swinging-bucket rotor for 17 h at 100,000 x g, 4 °C.
    5. Using a pipet, remove and discard the first 4.5 ml from the top of the gradient.
    6. Collect the next three volumes of 0.7 ml as individual samples.
    7. Transfer each sample to a fixed-angle ultracentrifuge tube. Bring each sample up to 3.5 ml using sterile, cold 20 mM Tris-HCl, pH 7.5.
    8. Centrifuge for 60 min at 100,000 x g, 4 °C.
    9. Decant the supernatant and resuspend and combine the three pellets in 3 ml of sterile, cold 20 mM Tris-HCl, pH 7.5.
    10. Centrifuge for 60 min at 100,000 x g.
    11. Decant the supernatant and remove excess buffer from the mouth of the tube using a Kim-wipe.
    12. Resuspend the pellet in 50 μl of sterile, cold 20 mM Tris-HCl.
      Notes: Purifying plant EVs requires a larger starting volume of apoplastic wash (~12-50 ml). A pale, milky pellet is visible when using larger volumes of apoplastic wash. However, a band is not visible in the discontinuous iodixanol gradient because the EVs are spread over a wider area.

Data analysis

Table 1 and Figure 5 show a typical data analysis for DiOC6 fluorescence, which includes the following steps:

  1. Average the three readings for each sample. Calculate the mean value for the blank (avg. blank) by determining the mean of the three sample means (i.e., the average of all nine blank readings).
  2. Subtract the mean value for the blank from the mean values for each positive control sample and each test sample.
  3. After subtracting the blank value, determine the mean value of the three positive control samples, and then divide each individual value by this mean (each should be close to 1.0). These numbers will be used to calculate the standard deviation of the positive control samples.
  4. Divide each of the test sample values (after subtracting the blank) by the mean of the positive control (this normalizes the test samples relative to the positive control).
  5. Determine the mean of these values to determine the average ratio of DIOC6 fluorescence in the test samples compared to the positive control.
  6. Calculate standard deviation and determine statistical significance using a two-tailed unpaired Student’s t-test.

    Table 1. Example of raw data from DiOC6 fluorescence quantification. The following data was generated from a replicate of an experiment presented in Rutter and Innes (2017). The experiment compared EV secretion between mock-treated Arabidopsis thaliana plants (Positive Control) and plants infected with a virulent strain of Pseudomonas syringae (Test).

    P value = 4.74 x 10-5


    Figure 5. Example graph of DiOC6 fluorescence. The above graph was generated from a replicate of an experiment presented in Rutter and Innes (2017). The experiment compared EV secretion between mock-treated Arabidopsis thaliana plants (Positive Control) and plants infected with a virulent strain of Pseudomonas syringae (Test).

    Note: Although DiOC6 staining provides an estimate of EV yield, it does not provide information on EV concentration and sizes. To determine these values, one must have access to a nanoparticle tracker, which, as the name implies, are instruments that can track individual particles in a solution. By monitoring the Brownian motion of individual EVs, these instruments can calculate EV sizes, and can count the number of EVs in a defined volume. There are currently two manufacturers of nanoparticle trackers, Particle Metrix (https://www.particle-metrix.de/en/products/zetaview-nanoparticle-tracking.html) and Malvern Instruments (http://www.malvern.com/en/products/technology/nanoparticle-tracking-analysis/).

Notes

  1. Because EV secretion is enhanced during stress, it is important to use healthy plants. It is also important to limit cytoplasmic contamination by avoiding damaged plants and handling the rosettes gently.
  2. We recommend following up total membrane quantification with an immunoblot for known plant EV markers, such as PEN1 or PATL1 (Rutter and Innes, 2017). Parallel increases or decreases in EV proteins bolster the DiOC6 data and help rule out the presence of other contaminating membranes.
  3. When isolating large volumes of apoplastic wash fluid, 1% plant PIC and 0.2 mM DPDS should be added immediately to wash samples to prevent degradation by apoplastic or cellular proteases.

Recipes

  1. 0.5x Murashige and Skoog agar (0.5 L)
    Dissolve 1.1 g Murashige Skoog basal salt mixture in 500 ml of deionized water
    Adjust pH to 5.7-5.9 with 1 N KOH
    Add 4 g (0.8%) agar (w/v)
    Autoclave at 121 °C for 20 min
    Store the medium at room temperature
    Poured plated can be stored at 4 °C
  2. Vesicle isolation buffer (VIB)
    20 mM MES hydrate
    2 mM CaCl2
    0.1 M NaCl
    Adjust pH to 6 with 10 N NaOH
    Autoclave at 121 °C for 20 min
    Note: To avoid possible contamination, use two sources of VIB. VIB for infiltrating plants is made in advance and stored at room temperature. VIB used for washing or resuspending vesicles is filtered through a 0.2 μm filter and stored at 4 °C.
  3. 20 mM Tris-HCl, pH 7.5
    1 M Tris stock, pH to 7.5 with concentrated HCl
    Dilute to 20 mM with distilled water
    Autoclave at 121 °C for 20 min
  4. DiOC6 staining solution
    10 μM DiOC6 diluted in sterile 20 mM Tris-HCl pH 7.5
    1% PIC (optional)
    0.2 mM DPDS (optional)
  5. Vesicle resuspension buffer
    Sterile 20 mM Tris-HCl pH 7.5
    1% PIC (optional)
    1.2 mM DPDS (optional)

Acknowledgments

This work was supported by a grant from the United States National Science Foundation (IOS-1645745) to R.W.I. A condensed version of this protocol was presented in Rutter and Innes (2017).

References

  1. An, Q., van Bel, A. J. and Huckelhoven, R. (2007). Do plant cells secrete exosomes derived from multivesicular bodies? Plant Signal Behav 2(1): 4-7.
  2. Colombo, M., Raposo, G. and Thery, C. (2014). Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30: 255-289.
  3. Davis, D. J., Kang, B. H., Heringer, A. S., Wilkop, T. E. and Drakakaki, G. (2016). Unconventional protein secretion in plants. Methods Mol Biol 1459: 47-63.
  4. Halperin, W. and Jensen, W. A. (1967). Ultrastructural changes during growth and embryogenesis in carrot cell cultures. J Ultrastruct Res 18(3): 428-443.
  5. Regente, M., Corti-Monzon, G., Maldonado, A. M., Pinedo, M., Jorrin, J. and de la Canal, L. (2009). Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins. FEBS Lett 583(20): 3363-3366.
  6. Rutter, B. D. and Innes, R. W. (2017). Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins. Plant Physiol 173(1): 728-741.

简介

细胞外囊泡(EVs)通过传递蛋白质和RNA在细胞间通讯中发挥重要作用。 虽然植物细胞分泌电动汽车,但是它们最近才被孤立,并且关于它们的生物发生,释放,摄取和功能的问题仍然没有得到回答。 在这里,我们提出了一个详细的方案,用于从拟南芥叶片的脱水洗涤中分离EV。 可以使用荧光染料定量分离的EV,以评估总膜含量。
【背景】细胞外囊泡(EVs)是介导蛋白质,脂质和遗传物质的细胞与细胞转移的膜结合结构。由于哺乳动物EVs转运RNA和调节免疫反应的能力,对哺乳动物EV的兴趣已经增长。哺乳动物EV通常被分离用于从培养细胞的培养基中研究,以及生物流体的增长列表(Colombo等,2014)。植物电动车也被认为在免疫反应中起作用,但比较缺乏(An et al。,2007; Davis et al。,2016)。这在很大程度上归因于没有孤立的方法。
  虽然植物EVs自1967年以来一直被观察到,使用透射电子显微镜,但直到2009年才开发出分离方法(Halperin和Jensen,1967)。 Regente等人(2009)从吸水向日葵(向日葵)种子中分离出小(直径50-200nm)小泡样结构。我们修改了Regente等人提出的方法(2009)从拟南芥玫瑰花的脱卵清洗中分离囊泡。为了确定哪些条件诱导或损害EV分泌,我们还设计了一种使用荧光亲脂性染料的3,3'-二己氧基亚硫氰酸碘(DIOC6(3))染色EV颗粒的方法。在没有复杂形式的纳米粒子跟踪的情况下,这种相对简单的方法可以量化总膜含量,并可用于间接测量电动汽车的浓度(Rutter and Innes,2017)。为了更精确的测量,并且为了评估EV的尺寸分布,可以使用纳米粒子跟踪。我们的方案能够研究植物EV含量和组成,以及介导EV生物发生和释放的途径和条件。

关键字:拟南芥, 细胞外囊泡, Evs, 质外体洗涤, DiOC6, 荧光定量

材料和试剂

  1. Micropore TM手术胶带(3M,目录号:1530-1)
  2. 透明塑料圆顶(悍马国际,目录号:11-3348)
  3. 30毫升无针注射器(BD,目录号:309650)
  4. 50ml锥形管(VWR,目录号:89039-656)
  5. 移液器提示
  6. 纸巾
  7. (Thermo Fisher Scientific,Thermo Scientific TM,目录号:3120-0250)的250ml塑料瓶(w / out cap)
  8. 15毫升锥形管(VWR,目录号:89039-668)
  9. 1.5ml微量离心管(VWR,目录号:20170-038)
  10. 10ml注射器
  11. 5毫升无针注射器(BD,目录号:309646)
  12. Acrodisc0.45μm注射器过滤器(Pall,目录号:4454)
    注意:如果关注细菌污染,也可以使用Acrodisc 0.22μm过滤器(Pall,目录号:4192)。
  13. Pryme PCR 8条0.2ml管(MIDSCI,目录号:AVSST)
  14. Kim-wipes(KCWW,Kimberly-Clark,目录号:34120)
  15. COSTAR EIA / RIA板,96孔,半面积,无盖,平底,未处理的黑色聚苯乙烯板(Corning,Costar ®,目录号:3694)
  16. 5.8 ml转移移液管(Thermo Fisher Scientific,目录号:222-1S)
  17. 培养皿,100 x 15毫米(VWR,目录号:25384-302)
  18. 拟南芥种子
  19. 漂白剂(奥斯汀,目录号:90000360)
  20. PRO-MIX PGX生物杀菌剂灌封混合物(Premier Tech Horticulture,目录号:10382RG)
  21. Optiprep TM密度梯度介质(Sigma-Aldrich,目录号:D1556)
  22. Murashige Skoog基础盐混合物(Sigma-Aldrich,目录号:M5524)
  23. 氢氧化钠(NaOH)颗粒(Avantor Performance Materials,MACRON,目录号:7708-10)
  24. 氢氧化钾(KOH)颗粒(Fisher Scientific,目录号:P250-500)
  25. 琼脂(Sigma-Aldrich,目录号:05040)
  26. MES水合物(Sigma-Aldrich,目录号:M8250)
  27. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  28. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  29. Trizma ®碱(Sigma-Aldrich,目录号:T1503)
  30. 盐酸(HCl)(EMD Millipore,目录号:HX0603-3)
  31. 3,3'-二己基氧羰基碳菁碘化物(DiOC 6(3))(Thermo Fisher Scientific,Invitrogen公司,目录号:D273)
  32. 植物蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)
  33. 2,2'-二吡啶基二硫化物(DPDS)(Sigma-Aldrich,目录号:149225)
  34. 0.5x Murashige和Skoog琼脂(见食谱)
  35. 囊泡隔离缓冲液(VIB)(见食谱)
  36. 20mM Tris-HCl,pH7.5(参见食谱)
  37. DiOC 染色溶液(参见食谱)
  38. 囊泡再悬浮缓冲液(见配方)

设备

  1. 金属镊子
  2. 剪刀
  3. 不锈钢镊子,3型(Techni-Tool,目录号:758TW474)
  4. 缩放
  5. 1升塑料烧杯
  6. 钛法国新闻咖啡壶,24 fl oz(雪峰,目录号:CS-111)
  7. 真空室,0.20 Cu。英尺。 (SP科学软件 - 贝尔艺术产品 - H-B仪器,目录号:F42031-0000)
  8. 真空泵,3.5 CFM(理想真空产品,目录号:P101532)
  9. JA-14固定角转子(Beckman Coulter,型号:JA-14,目录号:339247)
  10. 移液器
  11. MJ研究PTC-200热循环仪(MJ Research,目录号:8252-30-0001)
  12. 荧光酶标仪
    注意:我们使用Appliskan TM 荧光平板阅读器(Thermo Fisher Scientific,Thermo Scientific > TM ,目录号:5230000)以测量DiOC 6 检测多个波长可以工作。
  13. SW 41 Ti转子,摆动斗,钛,6×13.2ml,41,000rpm,288,000xg(Beckman Coulter,型号:SW 41 Ti,目录号:331362)
  14. 厚壁聚碳酸酯离心管(3.5ml,13×51mm)(Beckman Coulter,目录号:349622)
  15. Thinwall,Ultra-Clear TM 13.2ml,14×89mm超速离心管(Beckman Coulter,目录号:344059)
  16. TLA100.3固定角转子(Beckman Coulter,型号:TLA-100.3,目录号:349481)
  17. Avanti J-26S XP离心机(Beckman Coulter,型号:Avanti J-26S XPI,目录号:B22989)
  18. 荧光灯显微镜
  19. 生长室
  20. 冰桶(schuett-biotech,Spongex,目录号:3.680 052)
  21. Optima TLX超速离心机(Beckman Coulter,型号:Optima TM TLX,目录号:361545)
  22. 纳米粒子跟踪器(粒子Metrix,型号:ZetaView ®或Malvern Instruments,型号:Nanosight NS300)

程序

  1. 准备植物
    1. 用50%漂白剂灭活拟南芥种子不超过5分钟,用无菌dH 2 O洗3次。每次洗涤步骤应持续1-2分钟。
    2. 将种子放在含有0.8%琼脂的0.5x Murashige和Skoog(MS)培养基上(见食谱)。
    3. 将医疗胶带缠绕在每个MS板的周围,并将板在4°C的黑暗中储存2天
    4. 2天后,将板移至短时间条件(9小时,22℃,150μEm -2)。将板垂直放置,让种子发芽并生长7天
    5. 将幼苗转移到PRO-MIX PGX。用清洁的塑料圆顶覆盖幼苗一周。在第7天,破裂穹顶。第二天,完全取下穹顶。水每周两次或根据需要。允许植物在收获前总共生长6周。
    注意:首先在MS板上种植幼苗,然后将其转移到土壤中,确保每个实验的健康植物数量确切。每个基因型/治疗的典型实验需要约36株植物。我们已经发现,六周龄的植物产生最小量的细胞间清洗液,细胞污染风险最小。

  2. 准备用于脱发液洗涤收集的工具
    1. 使用在火焰中加热的金属镊子,在30ml注射器的末端融化8个附加孔(图1和视频1)。
      注意:这些额外的通道可以最佳地收集脱水洗涤液,并在主孔被堵塞时提供另外的路线。
    2. 使用加热的镊子多次穿透50ml锥形管帽,产生直径约2.5厘米的圆。打出圆圈,在帽子的中间创造一个洞。软化帽子在火焰中,并将其滑入30毫升注射器,直到距离顶部约1.5-2厘米。注射器现在可以拧紧到一个50ml锥形管上(图1和视频1)。
      注意:这样可以防止注射器的末端淹没在脱发液中。


      图1.用于脱髓鞘洗涤收集的工具。通过将整个拟南芥花粉包装在30ml注射器中来收集载脂蛋白洗涤液。另外八个孔用注射器熔化到注射器的末端,以产生用于洗涤物流过的通道。将一个2.5厘米的孔从50ml锥形管帽中冲出。然后将盖子滑到距离顶部2cm的注射器上。这样可将注射器牢固地拧入50ml锥形管中。见视频1.

      Video 1. EV isolation demonstration. This video demonstrates how to prepare the syringe and conical tube apparatus shown in Figure 3, and the procedures for extracting apoplastic wash fluid from leaves and subsequent purification of EVs from this fluid.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


  3. 收集脱屑液清洗
    注意:从图2中可以看出,从apoplastic洗涤收集到DiOC 6 定量的工作流程概述。


    图2.一般工作流程通过用等渗缓冲液真空浸润完全拟南芥玫瑰花结收集沉积物洗涤液,使用低速离心旋转洗涤渗透的缓冲液,并过滤洗涤以清除任何大的碎屑。然后使用差速离心法处理洗涤液以分离EV,其可以用亲脂性染料DiOC 6进行染色。在EV被洗涤并再次沉淀以除去多余的染料之后,可以测量EV颗粒中的DiOC <6>荧光以间接量化EV的数量。

    1. 使用剪刀切割每个拟南芥植物在根部。
      注意:离开一点根将有助于以后操纵玫瑰花结。请参阅视频1。
    2. 在1L塑料烧杯中收集收获的花瓣并记录质量。
      注意:每个治疗/基因型包括三个生物重复。重复应该是相同的体积,不应小于0.5毫升。需要约3 g玫瑰花凝胶来收集〜0.5 ml的脱脂洗液。
    3. 用水冲洗玫瑰花3次以除去土壤颗粒。
    4. 小心地将玫瑰花放在法国印刷机中,使用300-500毫升的囊泡隔离缓冲液(VIB)(参见食谱)。将盖子放在法国印刷机上,轻轻地放下柱塞,直到植物完全淹没
    5. 将法国压力机,植物和缓冲液放在真空室中。施加真空20秒。
    6. 从真空室中取出法国压力机,取下盖子,将VIB倒入1L塑料烧杯中。从法国新闻稿中删除玫瑰花形。
      注意:VIB可以在同一天重复使用多次,但是一旦用于真空渗透,VIB不应该被存储在另一天使用。
    7. 通过将根部轻轻摇动,然后用纸巾刷洗叶子,从植物中除去多余的VIB
    8. 将玫瑰花根部放在30 ml注射器开口的顶部(图3A)。轻轻地将玫瑰花絮制成注射器,不会破碎或撕裂叶子(图3B)。一旦花瓣足够远,将注射器轻轻敲入硬表面将迫使其完全进入注射器(图3C)。
      注意:莲座可以插入2或3.通常,4-6周的玫瑰花瓣将适合30毫升注射器内。玫瑰花不应该如此紧密地包装,以致损坏组织或阻止来自注射器的脱水酶洗涤液的流动。请参阅视频1。
    9. 将注射器拧入50ml锥形管中(图3D),并将管和注射器置于250ml塑料瓶中(图3E)。围绕注射器顶部固定的帽子应防止管子落入瓶子中。


      图3.包装玫瑰花蛋白用于脱卵清洗提取。 A.玫瑰花被放在注射器的顶部。将玫瑰花被轻轻注入注射器。 C.将注射器轻轻敲打在坚硬的表面上,以迫使玫瑰花入其中。 D.将注射器拧入50ml锥形管中。将玫瑰花环,注射器和50ml锥形管放入250ml塑料瓶中,然后将其放入JA-14转子中进行离心。见视频1.

    10. 将注射器,50ml锥形管和瓶子放入离心机转子的槽中,并以700 x g,4°C离心20分钟。
      注意:此步骤是为JA-14固定角转子设计的。
    11. 从离心机上取下每个注射器和锥形管,同时小心保持管的角度。拧下注射器,并从管底部取出载脂蛋白洗涤液。确保避免任何颗粒状土壤颗粒。将载脂蛋白洗液转移到无菌的15 ml锥形管中,并将样品保持在冰上
    12. 使用10ml注射器,通过0.45μm过滤器过滤掉载脂蛋白洗涤液,加入新的无菌15 ml锥形管中。
      注意:过滤步骤是为了清除大块碎屑而进行的预防措施。这个步骤可能导致脱落素洗涤的丢失,如果脱脂洗涤体积小(约500μl),可以跳过。在这些情况下,10,000 x g离心分离应足以清除任何碎屑。

  4. EV隔离和DiOC 6 染色
    1. 将每个体积的脱脂洗涤转移到超速离心管。使用冷冻VIB将每个管中的体积提高到管的最大体积。
    2. 以10,000 x g,4℃离心30分钟。
    3. 仔细地,使用移液管将上清液转移到新的超速离心管。
      注意:离心后保持管的位置,并小心使吸管远离可能造粒的碎屑。
    4. 在40,000 x g,4℃离心60分钟。
    5. 倾倒上清液,用吸管从管的开口除去任何多余的缓冲液。将沉淀物重悬于100微升100微摩尔DiOC 6(20mM Tris-HCl pH7.5)中(参见食谱)。如果将EVs用于免疫印迹后,在DiOC 6上,加入1%植物蛋白酶抑制剂混合物(PIC)和0.2mM DPDS至DiOC 6染色溶液(参见食谱)量化。
      注意:当使用小体积的脱脂洗液时,EV颗粒是不可见的。当使用大量的脱脂洗液(≥12ml)时,会出现薄而半透明的颗粒。为了有效地重悬颗粒,使用移液管反复洗涤含有颗粒的管的一侧,加入一半添加的DiOC 6 溶液。 / em>
    6. 将再悬浮的囊泡转移到PCR管中,并在热循环仪中于37℃加热10分钟。
    7. 将样品转移到新的干净的超速离心管中,并使用冷冻的20mM Tris-HCl pH 7.5将体积提高到管的最大体积。
    8. 在40,000 x g,4℃离心60分钟。
    9. 倾倒上清液,使用移液管,然后用Kim-wipe去除管的开口和上部区域的多余缓冲液。
      注意:滗析后立即擦拭剩余的缓冲液,同时离心管仍然倒置是重要的。仔细删除所有多余的缓冲液,确保每个沉淀物以相同的总体积重新悬浮。
    10. 将沉淀重悬于52μl囊泡重悬浮液(20mM Tris-HCl pH7.5,参见食谱)。
      注意:如果将EV用于免疫印迹,在DiOC 6之后,将1%植物蛋白酶抑制剂混合物(PIC)和0.2mM DPDS添加到囊泡再悬浮缓冲液中量化。

  5. 收集荧光数据
    1. 在黑色聚苯乙烯,96孔,半面,平底EIA / RIA板中的孔中加入每个样品50μl。
      注意:对于典型的实验,有三种样品:缓冲液的阴性对照(20mM Tris-HCl + 1%植物PIC和0.2mM DPDS)以计算背景信号,确定囊泡的阳性对照在模拟条件下或遗传野生型背景下的分泌,以及测试样品以确定治疗或突变对囊泡分泌的影响。如前所述,包括每个样本的三个生物重复。
    2. 将板插入荧光微量酶标仪。在485 nm处激发样品并记录535 nm的荧光发射。每板三张读数。
      注意:记录荧光后,样品对于许多不同的测定法仍然有用,包括用于透射电子显微镜,荧光显微镜和免疫印迹的染色。

  6. EV净化
    注意:差速离心产生可能含有质外体的其他成分的粗制EV制剂。 EV组合的深入研究需要进一步的净化。以下协议旨在使用不连续的碘克沙醇浓度梯度将EV与其他污染物分离。净化过程的概述可以在图4中看到。


    图4.用于纯化EV的工作流程将粗EV加载在不连续的碘克沙醇梯度之上,并以10万x g,4℃离心17小时。离心后,分离出三种含EV的级分。将这些级分洗涤并重新沉淀以除去碘克沙醇。然后将所有三个EV样品合并成单个管,洗涤并造粒,以产生一个纯化的EV样品
    1. 使用无菌的冷VIB将分离的粗制EV制剂的体积加至0.5ml。将样品放在冰上。
    2. 使用水溶液制备40%(3ml,v / v),20%(3ml,v / v),10%(3ml,v / v)和5%(2ml,v / v)的碘克沙醇溶液60%碘克沙醇(OptiPrep)和无菌,冷VIB。
    3. 使用转移吸管,将iodixanol溶液加入超速离心管中。仔细地将解决方案叠加在一起,从60%的解决方案开始,以5%的解决方案结束。最后,将0.5 ml的原油EV添加到梯度的顶部。
    4. 以10万x g,4°C在摆动转子中离心17小时。
    5. 使用移液器,从坡度顶部移除并丢弃前4.5 ml。
    6. 收集接下来的三个体积的0.7毫升作为个别样品。
    7. 将每个样品转移到固定角超离心管。使用无菌,冷的20mM Tris-HCl,pH7.5将每个样品加至3.5ml
    8. 以100,000 x g,4℃离心60分钟。
    9. 倾析上清液,并将三个沉淀重新悬浮并结合在3ml无菌,冷的20mM Tris-HCl,pH7.5中。
    10. 以100,000 x g离心60分钟。
    11. 使用Kim-wipe将上清液倾析并从管口中除去多余的缓冲液
    12. 将沉淀重悬于50μl无菌,冷的20mM Tris-HCl中 注意:净化植物EV需要较大的脱脂洗涤起始体积(〜12-50毫升)。当使用较大体积的脱卵清洗时,可以看到苍白的乳状颗粒。然而,由于电动汽车在更广泛的区域扩散,所以在不连续的碘克沙醇梯度中不可见带。

数据分析

表1和图5显示了DiOC 6&lt; 6&gt;荧光的典型数据分析,其包括以下步骤:

  1. 平均每个样本的三个读数。通过确定三个样本平均值(即所有九个空白读数的平均值)来计算空白的平均值(平均空白)。
  2. 从每个阳性对照样本和每个测试样本的平均值中减去空白的平均值。
  3. 减去空白值后,确定三个阳性对照样本的平均值,然后将每个单独的值除以该平均值(每个值应接近1.0)。这些数字将用于计算阳性对照样品的标准偏差。
  4. 将每个测试样本值(减去空白)除以阳性对照的平均值(相对于阳性对照标准化测试样本)。
  5. 确定这些值的平均值,以确定与阳性对照相比,测试样品中DIOC <6>荧光的平均比例。
  6. 计算标准偏差,并使用双尾不配对的Student's 测试来确定统计学显着性。

    Table 1. Example of raw data from DiOC6 fluorescence quantification. The following data was generated from a replicate of an experiment presented in Rutter and Innes (2017). The experiment compared EV secretion between mock-treated Arabidopsis thaliana plants (Positive Control) and plants infected with a virulent strain of Pseudomonas syringae (Test).

    P value = 4.74 x 10-5


    图5. DiOC 6 荧光的示例图。 上图是从Rutter和Innes(2017)提供的实验的复制生成的。实验比较了模拟处理的拟南芥植物(阳性对照)和感染了丁香假单胞菌的毒株(Test)的植物之间的EV分泌。

    注意:虽然DiOC 染色提供EV产量的估计,但它不提供关于EV浓度和尺寸的信息。为了确定这些值,人们必须能够访问纳米粒子跟踪器,顾名思义,它是可以跟踪解决方案中的单个粒子的工具。通过监控单个电动车的布朗运动,这些仪器可以计算电动汽车的大小,并可以计算定义音量的电动汽车的数量。目前有两个制造商的纳米粒子跟踪器,Particle Metrix( https://www.particle-metrix.de/en/products/zetaview-nanoparticle-tracking.html )和Malvern Instruments( http: //www.malvern.com/en/products/technology/nanoparticle-tracking-analysis /

笔记

  1. 因为在压力期间EV分泌增强,使用健康植物很重要。通过避免损坏的植物和温和地处理玫瑰花瓣来限制细胞质污染也是重要的。
  2. 我们建议使用已知植物EV标记(如PEN1或PATL1)(Rutter and Innes,2017)的免疫印迹跟踪总膜定量。 EV蛋白的平行增加或减少可增强二氧化碳分析数据,有助于排除其他污染膜的存在。
  3. 当分离大量的外源性洗涤液时,应立即加入1%植物PIC和0.2mM DPDS以洗涤样品,以防止由外质蛋白或细胞蛋白酶降解。

食谱

  1. 0.5x Murashige和Skoog琼脂(0.5升)
    将1.1克Murashige Skoog基础盐混合物溶解在500毫升去离子水中 用1N KOH调节pH至5.7-5.9 加入4 g(0.8%)琼脂(w / v)
    在121°C高压灭菌20分钟
    将介质储存在室温下
    镀层可以在4°C下储存
  2. 囊泡隔离缓冲液(VIB)
    20mM MES水合物
    2mM CaCl 2
    1.1 M NaCl
    用10N NaOH调节pH至6 在121°C高压灭菌20分钟
    注意:为避免可能的污染,请使用两个VIB源。渗透植物的VIB预先制备并在室温下储存。用于洗涤或重新悬浮囊泡的VIB通过0.2μm过滤器过滤并在4℃下储存。
  3. 20mM Tris-HCl,pH7.5
    1M Tris储存液,用浓HCl滴定至7.5 用蒸馏水稀释至20 mM 在121°C高压灭菌20分钟
  4. DiOC 染色溶液
    在无菌20mM Tris-HCl pH7.5中稀释的10μMDiOC 6 /
    1%PIC(可选)
    0.2 mM DPDS(可选)
  5. 囊泡再悬浮缓冲液
    无菌20mM Tris-HCl pH 7.5
    1%PIC(可选)
    1.2 mM DPDS(可选)

致谢

这项工作得到了美国国家科学基金会(IOS-1645745)授予R.W.I.的授权。 Rutter和Innes(2017)介绍了该协议的精简版本。

参考

  1. An,Q.,van Bel,AJ和Huckelhoven,R。(2007)。&nbsp; 植物细胞是否分泌来自多泡体的外来体?植物信号Behav 2(1):4-7。
  2. Colombo,M.,Raposo,G。和Thery,C。(2014)。外来体和其他细胞外囊泡的生物发生,分泌和细胞间相互作用。 Annu Rev Cell Dev Biol 30:255-289。
  3. Davis,DJ,Kang,BH,Heringer,AS,Wilkop,TE和Drakakaki,G.(2016)。植物中非常规的蛋白质分泌。 Methods Mol Biol 1459:47-63。
  4. Halperin,W.and Jensen,WA(1967)。&nbsp; 在胡萝卜细胞培养物中的生长和胚胎发生过程中的超微结构变化。超结构Res 18(3):428-443。
  5. Regente,M.,Corti-Monzon,G.,Maldonado,AM,Pinedo,M.,Jorrin,J.and de la Canal,L.(2009)。&nbsp; 向日葵胚胎性液体的囊泡部分与潜在的外来体标记蛋白相关。 FEBS Lett 583(20):3363-3366。
  6. Rutter,BD and Innes,RW(2017)。&nbsp; 细胞外 从叶片中分离的囊泡携带应激反应蛋白。植物生理学173(1):728-741。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rutter, B. D., Rutter, K. L. and Innes, R. W. (2017). Isolation and Quantification of Plant Extracellular Vesicles. Bio-protocol 7(17): e2533. DOI: 10.21769/BioProtoc.2533.
  2. Rutter, B. D. and Innes, R. W. (2017). Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins. Plant Physiol 173(1): 728-741.
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chuan shen
18792805578
Hi Brian,
I used DMSO to dilute DiOC6, because DiOC6 insoluble in Tris-Hcl, even though heating.
7/29/2018 6:56:40 PM Reply
Brian Rutter
Indiana University

That’s correct. The 10 mM stock of DiOC6 should be dissolved in DMSO. Then, dilute it in Tris-HCl to make a 100 uM solution.

7/29/2018 8:06:37 PM


chuan shen
18792805578
I apply the vacuum for 20 sec but I don't know whether -90 KPa is right, or maybe you can give me your advice. I want to know the difference between 4 ℃described in this protocol and 2 ℃ decribed in the paper "Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins. " What are the benefits of replacing MES (PH=6.0) with Tris-HCl (PH=7.5) in the paper? because I used Tris-HCl (PH=7.5) in my experiments and did't get expected results. I transfer the apoplastic wash to a sterile 2 ml conical tube because my apoplastic wash is small (~1000 μl), and the next 10,000 x g I used a normal high speed centrifuge instead of an ultracentrifuge,and the centrifuge tube is also a normal centrifuge tube, 40,000 x g centrifugation I used are all 6ml centrifuge tubes, because I think a small volume of liquid is sufficient with a small centrifuge tube, and no visible pellet after this step.
7/29/2018 3:13:27 AM Reply

Hi Chuan Shen,

Vacuum infiltration will very depending on the strength of you vacuum pump. I would say as long as your plants are being uniformly infiltrated and aren't leaking chloroplast into your wash fluids (as indicated by a green color) it's probably okay.

It shouldn't have an effect on your results whether you use 2*C or 4*C. You merely want to keep the EVs stable in a cool temperature while centrifuging.

I use MES (pH 6) when initially isolating EVs or handling them in general. This buffer is isotonic and was designed to match the pH of the apoplast, which is slightly acidic. I use Tris-HCl (pH 7.5) when staining with DiOC6, just in case an acidic pH affects DiOC6 fluorescence. The EVs are stable in either buffer.

I think your problem is probably in your technique and the equipment you're using during the ultracentrifuge steps. I use a tabletop ultracentrifuge and 3.5 ml thick wall, polycarbonate tubes with a fixed-angle rotor. I always make sure the tube contains its maximum volume. This prevents cracking of the tubes and washes away impurities. I also decant the supernatant. If you're using a swinging bucket rotor, this creates a loose pellet that can be easily lost. The shape of the tube can also affect the quality of your pellet, as well as pipetting out too much of the supernatant. Unfortunately, the pellet is invisible except when using very high amounts of apoplastic wash, so you'll have to guess where it is.

I would recommend going over you technique. Try looking at Thery et al. (2006). It has lots of helpful tips on technique for isolating EVs: Théry, Clotilde, et al. "Isolation and characterization of exosomes from cell culture supernatants and biological fluids." Current protocols in cell biology 30.1 (2006): 3-22.

If you're losing the majority of your EVs through your technique, you might also try using higher amounts of wash fluid.

Best of luck,
Brian

7/29/2018 10:03:03 AM


chuan shen
18792805578
Hello, I use this protocol to extract other plant leaf EVs, but low fluorescence value was collected. I find there are few differences between this protocol and the paper, so I want to ask for your help.
7/21/2018 9:15:49 AM Reply
Brian Rutter
Indiana University

Hi Chuan Shen,

I hope you got my email addressing you question.

I’d like to help you, but I need more information. There are lots of reasons why you may be getting low fluorescence. It could be that you aren’t collecting enough vesicles, and you need to use more apoplastic wash in you experiments. It could be that you are losing your pellet during the centrifugation steps by using a different kind of tube or pipetting technique. Maybe your fluorometer has a different level of sensitivity or your DIOC has gone bad.

If you share your protocol with me. Maybe I can see what could be the problem.

Best of luck,
Brian

7/20/2018 7:06:48 PM