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Jan 2018
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Quantitative Analysis of Cargo Density in Single-extracellular Vesicles by Imaging
成像技术定量分析单细胞外囊泡中内容物的密度   

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Abstract

Function of extracellular vesicles such as exosomes and microvesicles is determined by their wide ranges of cargoes inside them. Even in the pure exosomes or microvesicles the cargo contents are very heterogeneous. To understand this heterogeneous nature of extracellular vesicles, we need information of the vesicles, which will give us some parameters including vesicle size, number and cargo content of each vesicle. Here, we describe a new method to quantify cargo density in single-extracellular vesicles. Staining of extracellular vesicles in a membrane lipid content-proportionate manner and immobilization of extracellular vesicles onto glass substrate allow us to obtain cargo density information of single-extracellular vesicles. This protocol will be useful to analyze the effects of various drugs or genetic manipulation on vesicle generation and maturation including cargo sorting into heterogeneous extracellular vesicles.

Keywords: Extracellular vesicle (细胞外囊泡), Exosome (外泌体), Microvesicle (微囊泡), Cargo content (内容物含量), Cargo sorting (内容物分选), Single-extracellular vesicle (单细胞外囊泡), Imaging (成像)

Background

Extracellular vesicles are small membrane-bound vesicles released from a variety of cells, comprising exosomes and microvesicles based on the current knowledge of their biogenesis (van Niel et al., 2018). Extracellular vesicles contain various kinds of cargoes including cytosolic proteins, membrane proteins, lipids, microRNAs, and mRNAs. The cargoes are highly heterogeneous in their contents and variety depending on cell types. Extracellular vesicles play a role in erythrocyte maturation, antigen presentation, tumor metastasis, or prion disease and Alzheimer’s disease propagation, through cell-to-cell communication by carrying their cargoes from the donor to the recipient cells (Maas et al., 2017).

Exosomes are formed by fusion of multivesicular endosomes (MVEs) with the plasma membranes. On the other hand, microvesicles are formed by shedding of the plasma membranes. Endosomal sorting complexes required for transport (ESCRT) is known to be critical for the generation of lysosomal MVEs. It is reported that ceramide is important for the generation of exosomal MVEs (Trajkovic et al., 2008). The size of exosomes ranges from 30 nm to 100 nm, whereas that of microvesicles is from 50 nm to 1,000 nm (Karpman et al., 2017; van Niel et al., 2018). The mechanism of cargo sorting into extracellular vesicles, especially microvesicles, is still mysterious. For understanding the heterogeneous nature of extracellular vesicles, it is important to get more information on cargo density as well as the number and the size of single extracellular vesicles.

Conventionally, we can quantitate the amount of cargo in total extracellular vesicles obtained from culture media using western blot analysis. Since extracellular vesicles are highly heterogeneous as mentioned above, we need to get more precise figures of extracellular vesicles for the understanding of vesicle generation and maturation including the cargo sorting. We can solve this issue with detecting “cargo density of single-extracellular vesicles”. Here, we provide details on our new method for getting information of each extracellular vesicle with a cargo density. This protocol provides a useful readout of cargo content in each extracellular vesicle, and promote to unravel detailed molecular mechanism of the cargo sorting and accelerate clinical application of extracellular vesicles.

Materials and Reagents

  1. 10 μl, 200 μl, or 1,000 μl tips (Fukae-Kasei, Watson, catalog numbers: 123R-254CS, 123R-755CS, 110-706C)
  2. 200 μl gel loading tips (Quality Scientific Plastics, catalog number: 010-Q)
  3. 5 ml or 10 ml pipettes (Corning, catalog numbers: 4487, 4488)
  4. 50 ml conical centrifuge tubes (BD, Falcon, catalog number: 352070)
  5. 1.5 ml microcentrifuge tubes (Ina Optika, BIO-BIK, catalog number: CF-0150)
  6. 35 mm or 100 mm culture dish (Corning, catalog numbers: 430165, 430167) 
  7. 6-well plate (Corning, catalog number: 3516) 
  8. 35 mm glass bottom culture dish (MatTek, catalog number: P35G-0-14-C)
  9. 0.22 μm syringe filters (Millipore, catalog number: SLGVJ13SL)
  10. Kimwipes (Nippon Paper Crecias, catalog number: 62011)
  11. Aluminum foil (Mitsubishi Aluminum, Nippaku foil)
  12. Ultracentrifuge thick wall polycarbonate tubes (Beckman Coulter, catalog number: 349622)
  13. HeLa cells (RIKEN cell bank, catalog number: RCB0007)
  14. MDA-MB-231 cells (ATCC, catalog number: HTB-26)
  15. Dulbecco's modified Eagle medium (DMEM), high glucose, with L-Glutamine and Phenol Red (FUJIFILM Wako Pure Chemical, catalog number: 044-29765)
  16. Fetal bovine serum (FBS) (Biowest, catalog number: S1820)
  17. Penicillin-streptomycin Mixed Solution (P/S) (NACALAI TESQUE, catalog number: 09367-34)
  18. Trypsin-EDTA solution with phenol red (NACALAI TESQUE, catalog number: 32778-34) 
  19. FuGENE HD transfection reagent (Promega, catalog number: E2311)
  20. Opti-MEM, reduced serum medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 31985070)
  21. Extracellular vesicles-free fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: A2720803)
  22. Total exosome isolation reagent (from cell culture media) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 4478359)
  23. Paraformaldehyde (PFA) powder (Sigma-Aldrich, catalog number: P6148)
  24. Normal goat serum (Jackson Immuno Research, catalog number: 005-000-121)
  25. Lipophilic Tracer, DiD (Ex. 644 nm; Em. 665 nm) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D7757)
  26. Biotinyl Cap PE (PE-biotin) (Avanti Polar Lipids, catalog number: 870277)
  27. Biotin-conjugated bovine serum albumin (BSA-biotin) (Sigma-Aldrich, catalog number: A8549)
  28. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A8022)
  29. Streptavidin (Thermo Fisher Scientific, Invitrogen, catalog number: 434301)
  30. Antibody of cargo (e.g., anti-cMET antibody; Cell Signaling, catalog number: 3127S)
  31. Secondary antibody (e.g., anti-mouse IgG Alexa Fluor 594; Cell Signaling, catalog number: 8890S)
  32. Plasmid DNA of cargo (e.g., constructing plasmid DNA of GFP- or mCherry-fused cargo protein)
  33. Sodium chloride (NaCl) (NACALAI TESQUE, catalog number: 31319-45)
  34. Potassium chloride (KCl) (Wako, catalog number: 163-03545)
  35. di-Sodium hydrogenphosphate (Na2HPO4) (NACALAI TESQUE, catalog number: 31723-35)
  36. Potassium dihydrogenphosphate (KH2PO4) (NACALAI TESQUE, catalog number: 28721-55)
  37. NaOH (NACALAI TESQUE, catalog number: 31511-05)
  38. Phosphate-buffered saline (PBS) (see Recipes)
  39. 4% PFA (see Recipes)

Equipment

  1. Glass vial (Maruemu, catalog number: 5-115-02)
  2. Forceps
  3. Pipettes (Gilson, models: PIPETMAN P10, P20, P200, P1000)
  4. Pipetter (Drummond Scientific, model: Pipet-Aid XP)
  5. Polypropylene microtube storage rack, 80 positions
  6. Test tube rack (Sanwakaken, catalog number: SS30-10)
  7. Clean hood (NK System, model: VH-1300BH-2A)
  8. CO2 incubator (Forma Scientific, model: 3158)
  9. Confocal laser scanning microscope (Zeiss, model: LSM 510 Meta)
  10. Transmitted Light Microscope (Olympus, model: IMT-2)
  11. Ultracentrifuge (Beckman Coulter, model: Optima TL)
  12. Ultracentrifuge rotor (Beckman Coulter, model: TLA-100.3)
  13. Refrigerated microcentrifuge (Tomy Seiko, model: MRX-151)
  14. Vortex mixer (Scientific Industries, model: Vortex-Genie 2)
  15. Refrigerator, 4 °C
  16. Freezer, -20 °C
  17. Cold room, 4 °C

Software

  1. LSM510 v4.2 operating software (Zeiss)
  2. ImageJ (https://imagej.nih.gov/ij/)
  3. Excel for Mac 2011 (Microsoft)

Procedure

  1. Overall procedure
    Overall procedure for analyzing cargo density of single-extracellular vesicles is shown in Figure 1.


    Figure 1. Overall procedure for detecting cargo density in single-extracellular vesicles

  2. Production and purification of extracellular vesicles
    1. Cell culture and transfection for producing released extracellular vesicles
      1. HeLa cells or MDA-MB-231 cells are plated onto 35 mm culture dish or 6-well plate and maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 °C in 5% CO2
      2. When cells are at 70% confluence, remove the medium and wash the cells 2 times each with 2 ml of 37 °C DMEM without FBS and P/S.
      3. Replace medium with 1.2 ml of 37 °C DMEM containing 10% extracellular vesicles-free fetal bovine serum and 1% P/S.
      4. Incubate at 37 °C in 5% CO2 for 24 to 48 h. 
      Note: In case of analyzing the exogenous cargo (e.g., GFP- or mCherry-fused protein), transient transfection needs to be carried out using lipid-based transfection reagent (e.g., FuGENE HD) 24 to 48 h before the replacement with the extracellular vesicles-free medium.
    2. Purification of extracellular vesicles
      1. Flowchart of this part is shown in Figure 2.


        Figure 2. Flowchart of isolation of extracellular vesicles (exosomes and microvesicles) by differential centrifugation

      2. Cell culture media (1.2 ml) from 90%-95% confluent cells is collected in 1.5 ml microcentrifuge tube.
      3. Centrifuge at 500 x g at 4 °C for 10  min to remove the cell debris using a refrigerated microcentrifuge.
      4. Collect all 1.2 ml supernatant and transfer to a new 1.5 ml microcentrifuge tube.
      5. Centrifuge at 12,000 x g at 4 °C for 20  min using a refrigerated microcentrifuge to separate microvesicles and others.
      6. Collect the upper 1 ml of supernatant is directly purified by filtration through a 0.22 μm pore filter and transfer into the ultracentrifuge thick wall polycarbonate tube. Approximately 70 µl of sample is lost after the filtration.
      7. The pellet from Step B2e is diluted in 200 μl of phosphate-buffered saline (PBS) (Microvesicle-enriched fraction).
      8. Ultracentrifuge the filtrated supernatant using ultracentrifuge (Optima TL, rotor: TLA-100.3) at 100,000 x g at 4 °C for 70  min. 
      9. Remove supernatant (approximately 930 µl) carefully.
      10. Dilute the pellet in 200 μl of PBS (Exosome-enriched fraction).
      Note: Alternatively exosomes can be purified using total exosome isolation reagent according to the manufacturer's instructions.

  3. Preparation of functionalized glass surface
    1. Flowchart of this part is shown in Figure 3.


      Figure 3. Flowchart of preparation of functionalized glass surface

    2. Prepare BSA/BSA-biotin mixture with mixing 5 μl of 10 M BSA and 5 μl of 1 M BSA-biotin in 490 μl of PBS. 
    3. Cover the glass surface of glass bottom 35 mm culture dish with 400 μl of the BSA/BSA-biotin mixture.
    4. Incubate at room temperature for 20-30 min.
    5. Prepare 25 mM streptavidin solution by diluting 8 μl of 1 M streptavidin in 312 μl of PBS. 
    6. Remove excess BSA/BSA-biotin by gently rinsing the glass surface once with 1 ml of PBS.
    7. Cover the glass surface with 300 μl of the streptavidin solution.
    8. Incubate at room temperature for 10 min.
    9. Gently rinse the BSA/BSA-biotin/streptavidin-coated glass surface twice each with 1 ml of PBS.

  4. Labeling of extracellular vesicles
    1. Transfer the isolated extracellular vesicles (200 μl total) into a glass vial.
    2. Add 2 μl of 10 mg/ml DOPE-biotin into the glass vial and suspend to mix.
    3. Incubate at room temperature for 5 min.
    4. Add 2 μl of 1 mg/ml DiD into the glass vial and suspend to mix.
      Note: Because DiD staining is the most critical step for calculating the cargo density, it is important to take exact amount of DiD.
    5. Spin down the glass vial in a 50 ml conical tube at 200 x g at room temperature and take it out with forceps.
    6. Then incubate at room temperature for 15 min in the dark (Figure 4).


      Figure 4. Labeling of extracellular vesicles with DOPE-biotin and DiD in glass vial

  5. Immobilization of extracellular vesicles on glass surface
    1. The DOPE-biotin- and DiD-labeled extracellular vesicles are applied onto the functionalized glass surface using gel loading long tip (Figure 5).


      Figure 5. Immobilization of extracellular vesicles on functionalized glass surface. DOPE-biotin- and DiD-labeled extracellular vesicle solution is applied onto functionalized glass surface and made it flat against the surface tension.

    2. Leave to stand at 4 °C for 2-3 h in dark condition.
    3. Rinse twice each with 2 ml of PBS.
    Note: In case of analyzing the transiently expressed exogenous cargo (e.g., GFP- or mCherry-fused protein), skip to “(G) Imaging” immediately.

  6. Immunofluorescence staining of endogenous cargo in extracellular vesicles
    1. Fix immobilized extracellular vesicles with 4% PFA for 5 min in dark condition.
    2. Rinse extracellular vesicles twice with 2 ml of PBS and pipette out all solution.
    3. Add 150 μl of PBS containing 10% normal goat serum and incubate at room temperature for 30 min in dark condition.
    4. Rinse extracellular vesicles once with 2 ml of PBS and pipette out all solution.
    5. Add 150 μl of anti-cargo antibody (e.g., anti-cMet antibody) in PBS and incubate at room temperature for 60-90 min in dark condition.
    6. Rinse extracellular vesicles once with PBS and pipette out all solution.
    7. Add 150 μl of fluorescent-dye conjugated secondary antibody (e.g., Alexa 488 or Alexa 594) in PBS and incubate at room temperature for 30 min in dark condition.
    8. Rinse extracellular vesicles twice each with 2 ml of PBS.
    9. Store at 4 °C in dark condition, and observe in 2-3 days.

  7. Imaging of fluorescence-labeled cargo and DiD using fluorescence microscope
    Note: For this imaging part we will show about the exosome sample as a representative.
    1. Turn on the confocal laser scanning microscope (Zeiss, LSM 510 Meta).
    2. Open the Zeiss LSM510 v4.2 operating software.
    3. Turn on the appropriate laser (e.g., Argon laser for excitation of GFP or Alexa 488, 543 nm wavelength He-Ne laser for excitation of mCherry or Alexa 594, 633 nm wavelength He-Ne laser for excitation of DiD).
    4. Select 63x objective lens.
    5. Set the glass bottom 35 mm culture dish with immobilized and cargo-labeled extracellular vesicles on the specimen holder.
    6. Carefully find the glass surface using visible light under eyepiece viewing.
    7. Change the observation status to laser scanning.
    8. In the “Scan Control panel”, “Mode”, set the level of zoom to x1 (Default).
    9. In the “Scan Control panel”, “Channels”, set the pinhole to “200”. 
    10. Adjust the focus with DiD channel using “Fast XY”.
    11. Collect averaged images (e.g., number is selected to 4 for the average) of DiD and fluorescent cargo (e.g., GFP- or mCherry-fused cargo or immunostained cargo with anti-mouse IgG Alexa 488 or anti-mouse IgG Alexa 594 as secondary antibody) and save them as lsm format. 
    12. The acquired images of exosomes are shown in Figure 6 (in case of endogenous cargo) or in Kajimoto et al. (2013) or (2018) (in case of exogenous GFP- or mCherry-fused cargo).


      Figure 6. Fluorescent images of cargo (cMet-Alexa 594) and DiD in single-exosomes. Exosomes released from MDA-MB-231 cells are stained with anti-cMet antibody with Alexa 594 secondary antibody and DiD. Scale bars, 10 μm.

Data analysis

  1. Open the ImageJ software.
  2. Go to “Adjust” → “Set Measurements”, and check “Area”, “Mean gray value”, and “Integrated Density”, then click “Ok” button.
  3. Open the acquired images (lsm format file, here using the data of Figure 6 as example) with drag-and-drop to ImageJ icon (Figure 7A).
  4. Select DiD image (Channel 2) (Figure 7B).
  5. Set “Image” → “Type” → “8-bit” and confirm that the image is converted to greyscale (Figure 7C).
  6. Set “Edit” → “Invert” → “No (against the question “Process all 3 images?”)” for inverting the image of DiD (Figure 7D).
  7. Use “Image” → “Adjust” → “Threshold” to highlight the extracellular vesicles (Figure 7E). Basically using “Auto” would be working well. If necessary, adjust the threshold to the suitable level to count.
  8. Go to “Analyze” → “Analyze Particles”. There are some options in the “Analyze Particles” window (Figure 7F). Basically use default setting (Size: 0-Infinity, Circularity: 0.00-1.00, Show: Nothing) and just check “Display results” and “Add to Manager”. If there are too many small noises or you want to exclude particles based on size, adjust the numbers of “Size”. 


    Figure 7. Conversion of acquired images of cargo (cMet-Alexa 594) and DiD to be calculated with ImageJ

  9. In the “ROI Manager” window, go to “More” → “Multi Measure”, and just check “Measure All 2 Slices” and click “Ok” button.
  10. Get “Area”, “Mean gray value (Mean)”, and “Integrated Density (IntDen)” information of cargo (in channel 1) and DiD (in channel 2) of each extracellular vesicle (Figure 8).


    Figure 8. Results of “Area”, “Mean gray value (Mean)”, and “Integrated Density (IntDen)” of cargo (cMet-Alexa 594) and DiD in each exosome quantified by ImageJ. The total number of exosomes analyzed in this image is 122.

  11. Transfer the data sets to Excel by copy and paste.
  12. Obtain “Mean gray value (Mean)” information of background of cargo and DiD channels respectively.
    1. Open the acquired images again.
    2. Select vesicle-free dark area with “Rectangular” or “Oval” tool. 
    3. Go to “Analyze” → “Measure”, and get “Area”, “Mean gray value (Mean)”, and “Integrated Density (IntDen)” information of both cargo and DiD channels.
    4. Transfer the data sets to Excel with copy and paste.
  13. Calculate background of cargo and DiD of each extracellular vesicle respectively.
    Calculating formula: Background = (“Mean gray value [Mean]” of background) * (“Area” of extracellular vesicles)
    Note: If the calculating result becomes less than zero “0”, it would be fixed to zero “0”. This negative value means undetectable level of cargo or DiD.
  14. Calculate cargo density of each extracellular vesicle.
    Calculating formula:
    Cargo density = [(“Integrated Density (IntDen)” of cargo) - (Background of cargo)]/[(“Integrated Density (IntDen)” of DiD) - (Background of DiD)]
  15. Then make presentation diversely depending on the purpose of the study as in Kajimoto et al. (2013) or (2018).

Notes

  1. This protocol is applicable to all samples including adherent and non-adherent cells, plasma, and tissues. We used adherent cells as an example to describe the detailed protocol.
  2. The amount of extracellular vesicles varies in different cell types. In most cases, small-scale culture in 35 mm dishes or 6-well plates is enough, but in some cases, a large-scale culture in 60 mm or 100 mm dishes is required.
  3. If using the size- and density- dependent purification method for extracellular vesicles like differential centrifugation, it is important to use healthy cells for avoiding the contamination of apoptotic body or cell debris.
  4. Detailed conditions of Procedures C, D, and E are basically according to the previously reported fluorescence-based assay to measure single liposomes (Hatzakis et al., 2009).

Recipes

  1. Phosphate-buffered saline (PBS)
    1. Mix 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 18 mM KH2PO4 in 900 ml H2O
    2. Adjust to a final pH of 7.4
    3. Add H2O to 1,000 ml
    4. Autoclave to store (This is 10x PBS stock)
    5. Dilute 1:10 with H2O to make 1x PBS 
  2. 4% PFA
    1. Prepare 30 ml of H2O in 50 ml conical tube and heat to 60 °C
    2. Add 1.6 g of PFA powder in the 30 ml of H2O, 60 °C in the fume hood and mix well
    3. Add 4 μl of 10 N NaOH and mix well until the solution gets clear
    4. Add 4 ml of 10x PBS
    5. Add H2O to 40 ml and mix
    6. Cool, store at 4 °C in dark condition, and use in two weeks

Acknowledgments

This protocol was adapted from procedures published in Kajimoto et al. (2013) and (2018). This work was supported by grants from the JSPS KAKENHI, the Uehara Memorial Foundation, the Osaka Medical Research Foundation, and the Nakatani Foundation.

Competing interests

There are no conflicts of interest.

References

  1. Hatzakis, N. S., Bhatia, V. K., Larsen, J., Madsen, K. L., Bolinger, P. Y., Kunding, A. H., Castillo, J., Gether, U., Hedegard, P. and Stamou, D. (2009). How curved membranes recruit amphipathic helices and protein anchoring motifs. Nat Chem Biol 5(11): 835-841.
  2. Kajimoto, T., Okada, T., Miya, S., Zhang, L. and Nakamura, S. (2013). Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes. Nat Commun 4: 2712.
  3. Kajimoto, T., Mohamed, N. N. I., Badawy, S. M. M., Matovelo, S. A., Hirase, M., Nakamura, S., Yoshida, D., Okada, T., Ijuin, T. and Nakamura, S. I. (2018). Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes. J Biol Chem 293(1): 245-253.
  4. Karpman, D., Stahl, A. L. and Arvidsson, I. (2017). Extracellular vesicles in renal disease. Nat Rev Nephrol 13(9): 545-562.
  5. Maas, S. L. N., Breakefield, X. O. and Weaver, A. M. (2017). Extracellular vesicles: Unique intercellular delivery vehicles. Trends Cell Biol 27(3): 172-188.
  6. Trajkovic, K., Hsu, C., Chiantia, S., Rajendran, L., Wenzel, D., Wieland, F., Schwille, P., Brugger, B. and Simons, M. (2008). Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319(5867): 1244-1247.
  7. van Niel, G., D'Angelo, G. and Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19(4): 213-228.

简介

细胞外囊泡如外泌体和微泡的功能由其内部广泛的货物决定。 即使在纯外泌体或微泡中,货物内容也非常不均匀。 为了理解细胞外囊泡的这种异质性,我们需要囊泡的信息,这将给我们一些参数,包括囊泡大小,每个囊泡的数量和货物含量。 在这里,我们描述了一种量化单细胞外囊泡中货物密度的新方法。 以膜脂质含量 - 比例方式染色细胞外囊泡并将细胞外囊泡固定到玻璃基质上允许我们获得单细胞外囊泡的货物密度信息。 该方案将有助于分析各种药物或遗传操作对囊泡生成和成熟的影响,包括货物分选成异质细胞外囊泡。

【背景】细胞外囊泡是从各种细胞释放的小膜结合囊泡,包括基于其生物发生的当前知识的外来体和微泡(van Niel 等人,2018)。细胞外囊泡含有各种货物,包括胞质蛋白,膜蛋白,脂质,微小RNA和mRNA。根据细胞类型,货物的内容和种类高度异质。细胞外囊泡在红细胞成熟,抗原呈递,肿瘤转移或朊病毒病和阿尔茨海默病的传播中发挥作用,通过细胞与细胞之间的通信将其载体从供体携带至受体细胞(Maas 等。< / em>,2017)。

通过多囊泡内体(MVE)与质膜的融合形成外来体。另一方面,通过质膜的脱落形成微泡。已知转运所需的内体分选复合物(ESCRT)对于溶酶体MVE的产生是至关重要的。据报道,神经酰胺对外源性MVE的产生很重要(Trajkovic et al。,2008)。外泌体的大小范围为30 nm至100 nm,而微泡的大小为50 nm至1,000 nm(Karpman et al。,2017; van Niel et al。 ,2018年)。货物分选到细胞外囊泡,特别是微泡中的机制仍然是神秘的。为了解细胞外囊泡的异质性,重要的是获得关于货物密度以及单个细胞外囊泡的数量和大小的更多信息。

通常,我们可以使用蛋白质印迹分析来定量从培养基获得的总细胞外囊泡中的货物量。由于细胞外囊泡如上所述是高度异质的,我们需要获得更精确的细胞外囊泡图,以便了解囊泡的产生和成熟,包括货物分选。我们可以通过检测“单细胞外囊泡的货物密度”来解决这个问题。在这里,我们提供了我们的新方法的详细信息,以获得具有货物密度的每个细胞外囊泡的信息。该协议提供了每个细胞外囊泡中货物含量的有用读数,并促进解开货物分选的详细分子机制并加速细胞外囊泡的临床应用。

关键字:细胞外囊泡, 外泌体, 微囊泡, 内容物含量, 内容物分选, 单细胞外囊泡, 成像

材料和试剂

  1. 10μl,200μl或1,000μl吸头(Fukae-Kasei,Watson,目录号:123R-254CS,123R-755CS,110-706C)
  2. 200μl凝胶加载技巧(Quality Scientific Plastics,目录号:010-Q)
  3. 5毫升或10毫升移液器(康宁,目录号:4487,4488)
  4. 50毫升锥形离心管(BD,Falcon,目录号:352070)
  5. 1.5 ml微量离心管(Ina Optika,BIO-BIK,目录号:CF-0150)
  6. 35毫米或100毫米培养皿(康宁,目录号:430165,430167)&nbsp;
  7. 6孔板(康宁,目录号:3516)&nbsp;
  8. 35毫米玻璃底培养皿(MatTek,目录号:P35G-0-14-C)
  9. 0.22μm注射式过滤器(Millipore,目录号:SLGVJ13SL)
  10. Kimwipes(Nippon Paper Crecias,目录号:62011)
  11. 铝箔(三菱铝,Nippaku箔)
  12. 超速离心厚壁聚碳酸酯管(Beckman Coulter,目录号:349622)
  13. HeLa细胞(RIKEN细胞库,目录号:RCB0007)
  14. MDA-MB-231细胞(ATCC,目录号:HTB-26)
  15. Dulbecco的改良Eagle培养基(DMEM),高葡萄糖,含L-谷氨酰胺和酚红(FUJIFILM Wako Pure Chemical,目录号:044-29765)
  16. 胎牛血清(FBS)(Biowest,目录号:S1820)
  17. 青霉素 - 链霉素混合溶液(P / S)(NACALAI TESQUE,目录号:09367-34)
  18. 胰蛋白酶-EDTA溶液与酚红(NACALAI TESQUE,目录号:32778-34)&nbsp;
  19. FuGENE HD转染试剂(Promega,目录号:E2311)
  20. Opti-MEM,降低血清培养基(Thermo Fisher Scientific,Invitrogen TM ,目录号:31985070)
  21. 无细胞外囊泡的胎牛血清(Thermo Fisher Scientific,Gibco TM ,目录号:A2720803)
  22. 总外泌体分离试剂(来自细胞培养基)(Thermo Fisher Scientific,Invitrogen TM ,目录号:4478359)
  23. 多聚甲醛(PFA)粉末(Sigma-Aldrich,目录号:P6148)
  24. 正常山羊血清(Jackson Immuno Research,目录号:005-000-121)
  25. 亲脂示踪剂,DiD(Ex.644 nm; Em.665 nm)(Thermo Fisher Scientific,Invitrogen TM ,目录号:D7757)
  26. Biotinyl Cap PE(PE-biotin)(Avanti Polar Lipids,目录号:870277)
  27. 生物素结合的牛血清白蛋白(BSA-生物素)(Sigma-Aldrich,目录号:A8549)
  28. 牛血清白蛋白(BSA)(西格玛奥德里奇,目录号:A8022)
  29. 链霉抗生物素蛋白(Thermo Fisher Scientific,Invitrogen,目录号:434301)
  30. 货物抗体(例如,抗cMET抗体; Cell Signaling,目录号:3127S)
  31. 二抗(例如,抗小鼠IgG Alexa Fluor 594; Cell Signaling,目录号:8890S)
  32. 货物的质粒DNA(例如,构建GFP-或mCherry-融合货物蛋白的质粒DNA)
  33. 氯化钠(NaCl)(NACALAI TESQUE,目录号:31319-45)
  34. 氯化钾(KCl)(Wako,目录号:163-03545)
  35. 磷酸氢二钠(Na 2 HPO 4 )(NACALAI TESQUE,目录号:31723-35)
  36. 磷酸二氢钾(KH2 2 PO 4 )(NACALAI TESQUE,目录号:28721-55)
  37. NaOH(NACALAI TESQUE,目录号:31511-05)
  38. 磷酸盐缓冲盐水(PBS)(见食谱)
  39. 4%PFA(见食谱)

设备

  1. 玻璃小瓶(Maruemu,目录号:5-115-02)
  2. 钳子
  3. 移液器(Gilson,型号:PIPETMAN P10,P20,P200,P1000)
  4. 移液器(Drummond Scientific,型号:Pipet-Aid XP)
  5. 聚丙烯微管收纳架,80个位置
  6. 试管架(Sanwakaken,目录号:SS30-10)
  7. 清洁罩(NK系统,型号:VH-1300BH-2A)
  8. CO 2 培养箱(Forma Scientific,型号:3158)
  9. 共聚焦激光扫描显微镜(Zeiss,型号:LSM 510 Meta)
  10. 透射光显微镜(奥林巴斯,型号:IMT-2)
  11. 超速离心机(Beckman Coulter,型号:Optima TL)
  12. 超速离心机转子(Beckman Coulter,型号:TLA-100.3)
  13. 冷冻微量离心机(Tomy Seiko,型号:MRX-151)
  14. 涡旋混合器(科学工业,型号:Vortex-Genie 2)
  15. 冰箱,4°C
  16. -20°C冷藏柜
  17. 寒冷的房间,4°C

软件

  1. LSM510 v4.2操作软件(蔡司)
  2. ImageJ( https://imagej.nih.gov/ij/ )
  3. Excel for Mac 2011(微软)

程序

  1. 整体程序
    分析单细胞外囊泡货物密度的总体程序如图1所示。


    图1.检测单细胞外囊泡中货物密度的总体程序

  2. 细胞外囊泡的产生和纯化
    1. 细胞培养和转染产生释放的细胞外囊泡
      1. 将HeLa细胞或MDA-MB-231细胞接种到35mm培养皿或6孔板上,并保持在含有10%胎牛血清(FBS)和1%青霉素/链霉素(P / S)的Dulbecco改良Eagle培养基(DMEM)中。 )在37°C,5%CO 2 。&nbsp;
      2. 当细胞达到70%汇合时,取出培养基并用2ml不含FBS和P / S的37℃DMEM洗涤细胞2次。
      3. 用含有10%无细胞外囊泡的胎牛血清和1%P / S的1.2ml 37℃DMEM替换培养基。
      4. 在37°C,5%CO 2 中孵育24至48小时。&nbsp;
      注意:在分析外源性货物( 例如 ,GFP-或mCherry融合蛋白)的情况下,需要使用基于脂质的转染进行瞬时转染试剂( 例如 ,FuGENE HD)在用无细胞外囊泡的培养基替换前24至48小时。
    2. 细胞外囊泡的纯化
      1. 该部分的流程图如图2所示。


        图2.通过差速离心分离细胞外囊泡(外泌体和微泡)的流程图

      2. 将来自90%-95%汇合细胞的细胞培养基(1.2ml)收集在1.5ml微量离心管中。
      3. 使用冷冻微量离心机在4℃下以500×g离心10分钟离心以除去细胞碎片。
      4. 收集所有1.2ml上清液并转移至新的1.5ml微量离心管中。
      5. 使用冷冻微量离心机在4℃下以12,000 x g 离心20分钟以分离微泡等。
      6. 收集上层1ml上清液,通过0.22μm孔径过滤器过滤直接纯化,并转移到超速离心机厚壁聚碳酸酯管中。过滤后大约70μl样品丢失。
      7. 将来自步骤B2e的沉淀物稀释于200μl磷酸盐缓冲盐水(PBS)(富含微泡的部分)中。
      8. 使用超速离心机(Optima TL,转子:TLA-100.3)在100,000℃ x g 4℃下超滤离心过滤的上清液70分钟。&nbsp;
      9. 小心地去除上清液(约930μl)。
      10. 将沉淀物稀释在200μlPBS(富含外泌体的部分)中。
      注意:根据制造商的说明,可以使用总外泌体分离试剂纯化外泌体。

  3. 功能化玻璃表面的制备
    1. 该部分的流程图如图3所示

      图3.功能化玻璃表面的制备流程图

    2. 制备BSA / BSA-生物素混合物,在490μlPBS中混合5μl10M BSA和5μl1M BSA-生物素。&nbsp;
    3. 用400μlBSA/ BSA-生物素混合物覆盖玻璃底35mm培养皿的玻璃表面。
    4. 在室温下孵育20-30分钟。
    5. 通过在312μlPBS中稀释8μl1M链霉抗生物素蛋白制备25mM链霉抗生物素蛋白溶液。&nbsp;
    6. 用1 ml PBS轻轻冲洗玻璃表面一次,去除多余的BSA / BSA-生物素。
    7. 用300μl链霉抗生物素蛋白溶液覆盖玻璃表面。
    8. 在室温下孵育10分钟。
    9. 用1 ml PBS轻轻冲洗BSA / BSA-生物素/链霉抗生物素蛋白涂层玻璃表面两次。

  4. 细胞外囊泡的标记
    1. 将分离的细胞外囊泡(总共200μl)转移到玻璃小瓶中。
    2. 将2μl10mg/ ml DOPE-生物素加入玻璃小瓶中并悬浮混合。
    3. 在室温下孵育5分钟。
    4. 将2μl的1 mg / ml DiD加入玻璃瓶中并悬浮混合。
      注意:由于DiD染色是计算货物密度的最关键步骤,因此准确计算DiD值非常重要。
    5. 在室温下将玻璃瓶在50ml锥形管中在200 x g 下旋转,并用镊子将其取出。
    6. 然后在室温下在黑暗中孵育15分钟(图4)。


      图4.在玻璃瓶中用DOPE-生物素和DiD标记细胞外囊泡

  5. 细胞外囊泡在玻璃表面的固定化
    1. 使用凝胶加载长尖端将DOPE-生物素和DiD标记的细胞外囊泡施加到功能化玻璃表面上(图5)。


      图5.细胞外囊泡在功能化玻璃表面上的固定化。 DOPE-生物素和DiD标记的细胞外囊泡溶液应用于功能化玻璃表面,使其平坦抵抗表面张力。

    2. 在黑暗条件下,在4°C下静置2-3小时。
    3. 用2ml PBS冲洗两次。
    注意:如果分析瞬时表达的外源性货物( 例如 ,GFP-或mCherry融合蛋白),请立即跳至“(G)成像”。

  6. 细胞外囊泡内源性货物的免疫荧光染色
    1. 在黑暗条件下用4%PFA固定固定的细胞外囊泡5分钟。
    2. 用2ml PBS冲洗细胞外囊泡两次,并移出所有溶液。
    3. 加入150μl含有10%正常山羊血清的PBS,在室温下在黑暗条件下孵育30分钟。
    4. 用2ml PBS冲洗细胞外囊泡一次,并移出所有溶液。
    5. 在PBS中加入150μl抗货物抗体(例如,抗-cMet抗体),在室温下在黑暗条件下孵育60-90分钟。
    6. 用PBS冲洗细胞外囊泡一次并移出所有溶液。
    7. 在PBS中加入150μl荧光染料缀合的二抗(例如,Alexa 488或Alexa 594),并在室温下在黑暗条件下孵育30分钟。
    8. 用2ml PBS冲洗细胞外囊泡两次。
    9. 在4°C的黑暗条件下保存,并在2-3天内观察。

  7. 使用荧光显微镜成像荧光标记的货物和DiD
    注意:对于这个成像部分,我们将展示外泌体样本作为代表。
    1. 打开共聚焦激光扫描显微镜(Zeiss,LSM 510 Meta)。
    2. 打开Zeiss LSM510 v4.2操作软件。
    3. 打开适当的激光(例如,氩激光用于激发GFP或Alexa 488,543纳米波长He-Ne激光用于激发mCherry或Alexa 594,633 nm波长He-Ne激光用于激发DID)。
    4. 选择63x物镜。
    5. 将玻璃底部35mm培养皿与固定的和货物标记的细胞外囊泡放在样品架上。
    6. 在目镜观察下使用可见光仔细查找玻璃表面。
    7. 将观察状态更改为激光扫描。
    8. 在“扫描控制面板”,“模式”中,将缩放级别设置为x1(默认)。
    9. 在“扫描控制面板”,“通道”中,将针孔设置为“200”。&nbsp;
    10. 使用“快速XY”通过DiD通道调整焦距。
    11. 收集DiD和荧光货物(例如,GFP-或mCherry-融合货物或具有反 - 的免疫染色货物的平均图像(例如,数字选择为4的平均值)小鼠IgG Alexa 488或抗小鼠IgG Alexa 594作为二抗)并将其保存为lsm格式。&nbsp;
    12. 获得的外泌体图像显示在图6中(在内源性货物的情况下)或Kajimoto 等人(2013)或(2018)(在外源GFP-或mCherry-融合货物的情况下) 。


      图6.单外泌体中货物(cMet-Alexa 594)和DiD的荧光图像。 MDA-MB-231细胞释放的外泌体用抗cMet抗体染色,Alexa 594二抗和DiD 。比例尺,10μm。

数据分析

  1. 打开ImageJ软件。
  2. 转到“调整”→“设置测量”,选中“区域”,“平均灰度值”和“集成密度”,然后单击“确定”按钮。
  3. 打开采集的图像(lsm格式文件,此处使用图6中的数据作为示例),拖放到ImageJ图标(图7A)。
  4. 选择DiD图像(通道2)(图7B)。
  5. 设置“图像”→“类型”→“8位”并确认图像转换为灰度(图7C)。
  6. 设置“编辑”→“反转”→“否(针对问题”处理所有3个图像?“)”以反转DiD图像(图7D)。
  7. 使用“图像”→“调整”→“阈值”突出显示细胞外囊泡(图7E)。基本上使用“自动”将运作良好。如有必要,将阈值调整到适当的水平以进行计数。
  8. 转到“分析”→“分析粒子”。 “Analyze Particles”窗口中有一些选项(图7F)。基本上使用默认设置(大小:0-Infinity,圆度:0.00-1.00,显示:无),只需选中“显示结果”和“添加到管理器”。如果有太多小噪音,或者您想根据大小排除粒子,请调整“大小”的数量。&nbsp;


    图7.用ImageJ 计算获得的货物图像(cMet-Alexa 594)和DiD的转换

  9. 在“ROI Manager”窗口中,转到“More”→“Multi Measure”,然后选中“Measure All 2 Slices”并单击“Ok”按钮。
  10. 得到每个细胞外囊泡的货物(在通道1中)和DiD(在通道2中)的“面积”,“平均灰度值(平均值)”和“综合密度(IntDen)”信息(图8)。


    图8.通过ImageJ定量的每个外泌体中货物(cMet-Alexa 594)和DiD的“面积”,“平均灰度值(平均值)”和“综合密度(IntDen)”的结果。在该图像中分析的外泌体总数为122.

  11. 通过复制和粘贴将数据集传输到Excel。
  12. 分别获得货物和DiD通道背景的“平均灰度值(平均值)”信息。
    1. 再次打开采集的图像。
    2. 使用“矩形”或“椭圆形”工具选择无囊泡的暗区。&nbsp;
    3. 转到“分析”→“测量”,获得货物和DiD通道的“区域”,“平均灰度值(平均值)”和“综合密度(IntDen)”信息。
    4. 通过复制和粘贴将数据集传输到Excel。
  13. 分别计算每个细胞外囊泡的货物背景和DiD。
    计算公式:背景=(背景的“平均灰度值[平均值]”)*(细胞外囊泡的“面积”)
    注意:如果计算结果小于零“0”,则将其固定为零“0”。该负值意味着无法检测到货物或DiD。
  14. 计算每个细胞外囊泡的货物密度。
    计算公式:
    货物密度= [(货物的“综合密度(IntDen)”) - (货物背景)] / [(DiD的“综合密度(IntDen)” - ((DiD背景)]
  15. 然后根据研究目的进行不同的演示,如Kajimoto et al。(2013)或(2018)。

笔记

  1. 该方案适用于所有样品,包括贴壁细胞和非粘附细胞,血浆和组织。我们使用贴壁细胞作为例子来描述详细的方案。
  2. 细胞外囊泡的量在不同细胞类型中变化。在大多数情况下,35毫米培养皿或6孔培养皿中的小规模培养就足够了,但在某些情况下,需要在60毫米或100毫米培养皿中进行大规模培养。
  3. 如果使用尺寸和密度依赖的纯化方法进行细胞外囊泡如差速离心,重要的是使用健康细胞来避免细胞凋亡或细胞碎片的污染。
  4. 程序C,D和E的详细条件基本上是根据先前报道的基于荧光的测定单脂质体的测定(Hatzakis et al。,2009)。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    1. 混合1.37 M NaCl,27 mM KCl,100 mM Na 2 HPO 4 和18 mM KH 2 PO 4 在900毫升H 2 O中
    2. 调整至最终pH 7.4
    3. 将H 2 O加入1,000 ml
    4. 高压灭菌器存储(这是10倍PBS库存)
    5. 用H 2 O稀释1:10,制成1x PBS&nbsp;
  2. 4%PFA
    1. 在50ml锥形管中制备30ml H 2 O并加热至60℃
    2. 在通风橱中60℃的30ml H 2 O中加入1.6g PFA粉末并充分混合
    3. 加入4μl10N NaOH并充分混合直至溶液澄清
    4. 加入4毫升10倍PBS
    5. 将H 2 O加入40ml并混合
    6. 冷却,在4°C的黑暗条件下储存,并在两周内使用

致谢

该方案改编自Kajimoto et al。(2013)和(2018)中公布的程序。这项工作得到了JSPS KAKENHI,上原纪念基金会,大阪医学研究基金会和中谷基金会的资助。

利益争夺

没有利益上的冲突。

参考

  1. Hatzakis,N.S。,Bhatia,V。K.,Larsen,J.,Madsen,K.L。,Bolinger,P。Y.,Kunding,A。H.,Castillo,J.,Gether,U。,Hedegard,P。和Stamou,D。(2009)。 弯曲膜如何招募两亲性螺旋和蛋白质锚定基序。 Nat Chem Biol 5(11):835-841。
  2. Kajimoto,T.,Okada,T.,Miya,S.,Zhang,L。和Nakamura,S。(2013)。 正在进行的鞘氨醇1-磷酸受体激活介导外泌体多囊泡内体的成熟。 Nat Commun 4:2712。
  3. Kajimoto,T.,Mohamed,N.N.I。,Badawy,S.M.M.,Matovelo,S.A.,Hirase,M.,Nakamura,S.,Yoshida,D.,Okada,T.,Ijuin,T。和Nakamura,S.I。(2018)。 在F-肌动蛋白形成和货物的多泡内体上参与Gi蛋白的Gbetagamma亚基与S1P受体的参与分类到外泌体中。 J Biol Chem 293(1):245-253。
  4. Karpman,D.,Stahl,A。L.和Arvidsson,I。(2017)。 肾脏疾病中的细胞外囊泡。 Nat Rev Nephrol 13 (9):545-562。
  5. Maas,S.L。N.,Breakefield,X。O.和Weaver,A。M.(2017)。 细胞外囊泡:独特的细胞间递送载体。 Trends Cell Biol 27(3):172-188。
  6. Trajkovic,K.,Hsu,C.,Chiantia,S.,Rajendran,L.,Wenzel,D.,Wieland,F.,Schwille,P.,Brugger,B。和Simons,M。(2008)。 神经酰胺引发外泌体囊泡出现在多囊泡内体中。 Science 319(5867):1244-1247。
  7. van Niel,G.,D'Angelo,G。和Raposo,G。(2018)。 揭示细胞外囊泡的细胞生物学。 Nat Rev Mol Cell Biol 19(4):213-228。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kajimoto, T. and Nakamura, S. (2018). Quantitative Analysis of Cargo Density in Single-extracellular Vesicles by Imaging. Bio-protocol 8(24): e3111. DOI: 10.21769/BioProtoc.3111.
  2. Kajimoto, T., Mohamed, N. N. I., Badawy, S. M. M., Matovelo, S. A., Hirase, M., Nakamura, S., Yoshida, D., Okada, T., Ijuin, T. and Nakamura, S. I. (2018). Involvement of Gbetagamma subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes. J Biol Chem 293(1): 245-253.
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Feixiao Hu
Huazhong Agricultural University
Exosomes are formed by fusion of multivesicular endosomes (MVEs) with the plasma membranes. On the other hand, microvesicles are formed by shedding of the plasma membranes. Endosomal sorting complexes required for transport (ESCRT) is known to be critical for the generation of lysosomal MVEs. It is reported that ceramide is important for the generation of exosomal MVEs (Trajkovic et al., 2008).
2022/4/18 17:14:16 回复