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Oct 2019

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Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation
用柠檬酸钠和差速超速离心分离牛乳中多个含外泌体和膜囊泡的胞外小泡亚群   

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Abstract

Milk is a complex fluid that contains various types of proteins and extracellular vesicles (EVs). Some proteins can mingle with EVs, and interfere with their isolation. Among these proteins, caseins form micelles of a size comparable to milk EVs, and can thus be co-isolated with EVs. Preliminary steps that affect milk are crucial for EV isolation and impact the purity and abundance of isolated EVs. In the course of our previous works on cow’s milk EVs, we found that sodium citrate (1% final), which is a biocompatible reagent capable of breaking down casein micelles into 40-nm monomers, allowed the isolation of high quantities of EVs with low coprecipitation of caseins or other contaminating proteins. Using this protocol, we successfully separated different EV subsets, characterized in depth their morphology, protein content and small RNA enrichment patterns. We were also able to describe their biological function in a mouse model of intestinal inflammation. We, hereby, detail the differential ultracentrifugation procedure that leads to high quantify, medium specificity, isolation of different milk EV subsets from the same sample. More specifically, we highlight the use of sodium citrate as a standardized approach to isolate and study milk EVs and its potential for isolation techniques other than differential ultracentrifugation.

Keywords: Extracellular Vesicles (细胞外基质), EVs (Evs), Milk (牛乳), Exosomes (外泌体), Microvesicles (细胞微泡), Ultracentrifugation (超速离心), Bovine Milk (牛乳), Casein (酪蛋白), Citrate (柠檬酸盐)

Background

In our previous publications (Benmoussa et al., 2016, 2017, 2019b and 2019c; Benmoussa and Provost, 2019), we highlighted that bovine milk is a complex fluid containing a myriad of extracellular vesicles (EVs) subsets. Among these, exosomes are ~100-nm vesicles released when multivesicular bodies (MVB) fuse with the cell membrane. When subjected to ultracentrifugation, these sediment at centrifugation speeds equal or higher than 100,000 x g (P100K, where P stands for pellet) (Pieters et al., 2015). Other non-exosome EV subsets are found in milk and are comparable to exosomes, in shape and size, but sediments at a lower speed (e.g., 12,000 x g, P35K; 35,000 x g, P35K; 70,000 x g, P100K). These are thought to be originating from budding of the cell membrane and contain specific proteins and microRNAs different from exosomes content (Benmoussa et al., 2016, 2017, 2019b and 2019c; Benmoussa and Provost, 2019).

For a long time, separating these different milk EVs subsets remained challenging because of their morphological and chemical similarities. Moreover, protocols used in milk EV studies were often centered on the isolation of milk exosomes specifically, leading to the discarding of the biologically active non-exosome milk EVs (Benmoussa et al., 2019a, 2019b and 2019c).

When using differential ultracentrifugation to isolate milk EVs, the first steps are often impaired by the co-precipitation of milk proteins with EVs, which form a dense jelly at the bottom of the tubes (Zonneveld et al., 2014). Such jelly is formed when caseins are subjected to high mechanical pressures (Famelart et al., 1998; Zonneveld et al., 2014). Because this jelly is so dense, and practically impossible to resuspend, it is not clear whether some EV subsets are trapped within its matrix and some protocols recommend discarding this casein-rich jelly.

Certain reports suggested the use of density gradients or cushions to keep the EVs from mixing with the casein jelly (Zonneveld et al., 2014). Others suggested avoiding the formation of such jelly by discarding caseins before subjecting milk “serum” or whey to differential ultracentrifugation. To this end, milk is often mixed with acids (Somiya et al., 2018) or with cold EDTA to precipitate the caseins prior to milk EVs sedimentation (Wolf et al., 2015). However, such preprocessing of biological fluids have an immense impact on the isolation, quality and yield of extracellular vesicles (EVs) (Zonneveld et al., 2014). It also requires to discard low speed-pelleting EVs (P12K or P35K). In addition, there is a lack of information about the effect of acidification, or casein precipitation, on milk EVs, and the possibility that some EVs might be lost along the process.

This is especially important because there are multiple EV subsets in milk (Benmoussa et al., 2017 and 2019b) and because milk whey has a different microRNA and protein content than milk (Benmoussa et al., 2019b; Benmoussa and Provost, 2019), which, as microRNA are found within EVs, suggests the loss of certain EVs during casein precipitation. This is also supported by the discovery of microRNAs in milk-derived casein-rich products, like cheese (Benmoussa and Provost, 2019). Therefore, the biological activity of the milk EVs, and the bioactive molecules they transport, depend highly on the reagents/preprocessing steps and isolation protocols this fluid is exposed or subjected to (Zonneveld et al., 2014).

When we encountered this issue, we experimented different venues to avoid precipitating milk caseins while ensuring the separation of different structurally conserved, and functional, milk EV subsets. In milk, casein proteins are arranged in the form of micelles that are 70 to 200 nm in diameter, which is close to the size of certain EV subsets (Blans et al., 2017). Knowing that calcium is important for maintaining these micelles (de Kort et al., 2009, 2011 and 2012; Kort et al., 2012), we supposed that calcium chelation would prevent the formation of casein superstructures that hamper EV isolation by ultracentrifugation.

Several calcium-chelating agents have been previously investigated for their ability to disrupt casein micelles, including disodium uridine monophosphate (Na2UMP), disodium phosphate (Na2HPO4), trisodium citrate (referred to simply as "sodium citrate"), sodium phytate, sodium hexametaphosphate (SHMP) or ethylenediaminetetraacetic acid (EDTA) (Ward et al., 1997; de Kort et al., 2009, 2011 and 2012; Kort et al., 2012). These chelating agents interact with colloidal calcium phosphate (CCP) leading to the breaking of casein micelles into small monomers. This process augments the stability of milk during heating and change its physical properties, often reducing its viscosity (de Kort et al., 2009, 2011 and 2012; Kort et al., 2012).

Among these, sodium citrate is a biocompatible compound known for 100+ years for its ability to prevent casein curdling in the stomach of infants (Poynton, 1904). It is widely used to preserve biological fluids, like blood (Janse van Rensburg and van der Merwe, 2017), as a food additive and as the major form of rehydration salt recommended by the World Health Organization (Pizarro et al., 1986; Banipal et al., 2016). Notably, it was previously used to disrupt casein micelles to facilitate the isolation of milk fat globules (MFGs) by diafiltration and prevented pore clogging (Phan et al., 2014). Interestingly, sodium citrate specifically impacts milk protein gel formation upon high mechanical pressure, although the underlying mechanisms remain unclear, except for its dependence on the solution’s pH (Famelart et al., 1998).

In the course of our experimentations with sodium citrate, we found that pre-treating milk with sodium citrate (1% final) was a cost-effective way to avoid casein jellification and isolation of high quantities of different milk EVs using differential ultracentrifugation. We hereby detail the methods underlying this method that led to the discovery (Benmoussa et al., 2016 and 2017) and characterization (Benmoussa et al., 2017, 2019b and 2019c) of different EVs subsets in milk, keeping them functional and able to transfer their content to human cells (Benmoussa et al., 2019c). EVs, including exosomes, isolated by this methodology were able to modulate intestinal inflammation during experimental colitis (Benmoussa et al., 2019a).

Materials and Reagents

  1. Sterilized glass bottle (Pyrex, Corning Life-Science, catalog number: 1395-1L) or sterile plastic bottles for mixing milk and sodium citrate (Corning, catalog number: 431533)
  2. Falcon tubes 50 ml (any product, as long as sterile and pyrogen-free, e.g., Corning, catalog number: 352070, Fisher Scientific, catalog number: 14-432-22)
  3. 1.7 ml snap cap tubes (any product as long as sterile and pyrogen-free, e.g., Corning, Costar via Sigma-Aldrich, catalog number: CLS3620)
  4. 0.22 µm membrane microfilters (Corning, Sigma-Aldrich, catalog number: CLS431224)
  5. Serological pipets different volumes (any product as long as sterile and pyrogen-free, e.g., Sigma-Aldrich, catalog number: SIAL1485)
  6. Filtered 1 ml pipet tips (any product as long as sterile, pyrogen free and filtered, e.g., Thermo Scientific, catalog number: 94052410)
  7. Nitrile disposable gloves (any type)
  8. Sterile wipes/gauze (any as long as sterile single-packed, e.g., VWR, catalog number: CA95041-740)
  9. Optional: EDTA. Can be bought as ready-to-use solution (Sigma-Millipore, catalog number: 324506) or homemade from powdered EDTA
  10. Milk
    Use commercially available milk bought on the day of the experiment or raw cow milk collected and stored in “sterile conditions” (as sterile as possible using sterilized bottles and keeping the bottles closed until getting under a biological hood). In our work we used mostly pasteurized commercial ultrafiltered skim milk (Lactantia PureFilter, bought in a local grocery store) as it avoids creaming steps that could lead to variability. We recommend working with a pool of three milks with different preemption dates. Milk must be stored at 4 °C.
    Note: If working with raw milk, use pool from different cows to minimize interindividual differences. Noncommercial raw or pasteurized fresh cow milk can be frozen at -80 °C to avoid bacterial contamination/proliferation but note such storage might impact EVs content. If freezing milk, make sure it is previously skimmed and decellularized to minimize EVs formations due to milk fat globule or cell destruction upon freeze/thawing cycles. Freeze milk in small aliquots (50 ml) so, when thawing, it reaches melting point quicker and limit EVs destruction.
  11. Bradford's reagent (Sigma-Aldrich, catalog number: B6916)
  12. BCA Protein Assay Kit (Millipore, catalog number: 71285-M)
  13. Milli-Q water or any filtered ultra-pure water (e.g., Millipore, water SystemClear Sorting & Filtering, catalog number: ZRXQ003WW)
  14. NaCl (preferably low in endotoxins, e.g., Millipore-Sigma, SAFC, catalog number: 1.16224)
  15. KCl (e.g., Sigma-Aldrich, catalog number: P3911)
  16. Na2HPO4 (e.g., Sigma-Aldrich, catalog number: NIST2186II)
  17. KH2PO4 (e.g., Sigma-Aldrich, catalog number: NIST200B)
  18. HCl (e.g., Sigma-Aldrich, catalog number: 320331)
  19. EDTA (e.g., Sigma-Aldrich, catalog number: EDS-500G)
  20. NaOH solution in Milli-Q water (e.g., Sigma-Aldrich, catalog number: S8045)
  21. Sodium citrate dihydrate (Sigma-Aldrich, catalog number: W302600)
  22. Phosphate buffered saline
    Either bought as commercially available as filtered sterile phosphate buffered saline (PBS) pH 7.4 (Sigma-Aldrich, catalog number: P5493) or homemade.
  23. 2% sodium citrate solution (1 L) (see Recipes)
  24. Phosphate buffer saline (PBS) 10x (1 L) (see Recipes)
  25. EDTA 0.5 M recipe (100 ml) (see Recipes)

Equipment

  1. Tubes for ultracentrifugation compatible with rotor bellow (36 ml, Beckman, catalog number: 355631 or 344058 or other, see rotor compatibility)
  2. Ultracentrifuge Sorvall WX ultracentrifuge (or one of Thermo ScientificTM SorvallTM WX ultraCentrifuges, catalog numbers: 75000100, 7500090, 7500080)
  3. Rotor SureSpin 630 swinging bucket Rotor (Thermo Scientific, catalog number: 79368)
    Note: You can use another centrifuge and another rotor but make sure to get comparable material. Note that it is not recommended to use fixed-angle rotors as they will induce higher EV degradation.
  4. Pipetting device (any type, e.g., Drummond Scientific Portable Pipet-Aid XP via Mandel, catalog number: DRU-4-000-101)
  5. Orbital shaker (any type, e.g., Bel-ArtTM SP SciencewareTM SpindriveTM Orbital Shaker Platform, via Fisher-scientific, catalog number: 1451176)
  6. Rotating mixer/tube revolver (any type, e.g., Thermo-scientific, catalog number: 88881001)
  7. High precision balance (any type, e.g., Sartorius, catalog number: PRACTUM224-1S)
  8. Tube rack holder different sizes (any product)
  9. Laminar flow hood (if working on sterile conditions) (any product)
  10. Micropipettes, different volumes (any type as long as they are precise and pipetting is smooth enough to avoid too fast ejection of suspension liquid, e.g., PIPETMAN L P1000L, 100-1,000 µl, Metal Ejector, Gilson, catalog number: FA10006M)
  11. -80 °C freezer
  12. Magnetic stirrer

Procedure

Note: All procedure should be performed at 4 °C.

  1. Start ultracentrifuge and set device temperature at 4 °C.
  2. Mix 125 ml of skimmed milk and 2% sodium citrate 1:1 in a sterile glass or plastic bottle.
    Note: Pour sodium citrate first then milk to accelerate casein micelles breaking.
  3. Keep on a rocker for 15 min at 4 °C. Make sure milk is gently mixing (1/4th max speed).
    Note: This can be done in cold room or by putting ice on the rocker and milk on the ice.
  4. After 15 min, milk should clarify (get translucid with an aspect comparable to blood plasma) and ready for loading into ultracentrifugation tubes (Figure 1).


    Figure 1. Mixing skimmed milk with 2% sodium citrate 1:1 leads to milk clarification after 15 min incubation on a rocker at 4 °C
    Notes:
    1. Adding calcium carbonate reverses the clarification confirming the involvement of calcium chelation in the process.
    2. If the milk you use is not clarified, you can increase sodium citrate concentration to 3% or 4%.

  5. Fill the ultra-clear centrifuge tubes (Figure 2) completely with milk-sodium citrate mix.
    Note: Make sure the tubes are filled completely or they might break upon centrifugation.
  6. Put tubes in rotor tube holders and use sterile pipet to equilibrate mass between opposed tubes (Figure 2) by transferring milk-sodium-citrate mix between the opposed tubes (keeping comparable added mass of the tube, milk-citrate solution, tube holder and cap).


    Figure 2. Ultraclear thick ultracentrifugation tubes should be loaded into the tube holder before mass equilibration for ultracentrifugation

  7. Close caps and place them in the socket they are meant for (indicated by the number on the tube holder and the rotor, Figure 3).


    Figure 3. Ensure loading SureSpin tubes in the right socket to avoid equilibrium troubles

  8. Spin EVs are the desired speed. In our protocol we isolated low-speed sedimenting EVs at 35,000 x g (35K) for 2 h, at 4 °C. We used Sorvall WX TL-100 ultracentrifuge’s automated calculations of the K factors and set acceleration at A = 9 and break at D = 9.
    Note: If working with another ultracentrifuge, make sure to translate the g-force speed into the proper rotation per minute (RPM) considering rotor angle and k factor. If wishing to use different speeds, refer to the rotor and centrifuge manufacturer recommendations. If you work with raw milk, you can decellularize the liquid with two centrifugations at 1,000 x g for 10 min and 4,500 x g for 30 min, at 4 °C. Afterwards, you can proceed as described above.
  9. After 2 h, EVs should have sedimented forming a translucid gelatinous pellet on the bottom of the tube (Figure 4C).


    Figure 4. Non-diluted and PBS-diluted milk led to the formation of casein jelly after 35,000 x g ultracentrifugation. Dilution with sodium citrate leads to small EV-rich pellet with very little casein contamination. A. 35,000 x g pellet from non-treated milk. B. 35,000 x g pellet from diluted milk 1:1 in PBS. C. 35,000 x g pellet from diluted milk 1:1 in 2% sodium citrate contains very little contaminating casein.
    Note: The pellet on the right contains P35K EVs. It might not be visible prior to discarding the supernatant and suspension. It is well fixed to the bottom of the tube so there is little risk to lose it in the next steps.

  10. Transfer the supernatant into new clean/sterile ultracentrifugation tubes.
    Note: This can be done using a serological pipet and pipetting device or directly by pouring the supernatant into a new tube, this depending on the desired sterility levels.
  11. Equilibrate the supernatant tubes and centrifuge at 70,000 x g (70K) for 1 h, at 4 °C following the same procedure as previously.
  12. During the 70K ultracentrifugation, revert the remaining liquid free 35K pellet-containing tubes on a tube holder with sterile gauze/paper on the bottom of it, with the opening of the tubes touching the gauze/paper to allow remaining liquid to be discarded by capillarity within the gauze/paper (Figure 5).


    Figure 5. Reverting tubes over a paper sheet allows remaining liquid to be discarded by capilarity
    Note: This is important to avoid contamination with proteins from the supernatant and to keep precise suspension volumes. You can use pipet and tips to discard any remaining liquid or sterile gauze/paper inside the tube.

  13. Suspend all the pellets (6 tubes) in the same 1 ml sterile filtered PBS solution.
    Notes:
    1. The pellet is gelatinous and not easy to suspend. Avoid breaking it with the tips. Use pipet and 1 ml filtered tip to drop PBS on it and use PBS to gently “erode” the pellet starting from its sides and getting gently to the center. Try to be quick and efficient to avoid drying of the pellets but avoid rough mixing or you will end up with a foam limiting pellet suspension.
    2. You can also pour 100 μl PBS on the tubes you are not suspending while using 500 μl PBS to suspend the first tube. Transfer the 500 μl to the second tube having thus 600 μl for the second pellet, and so on. Depending on your application, you can include 0.5% EDTA to the PBS to help with the suspension with caution if planning on doing qPCR or for functional studies.
  14. Transfer suspended P35K EVs into a 1.7 ml Eppendorf tube and put on a rotating mixer at 4 °C at least for 24 h to allow full suspension of P35K EVs. EVs can then be stored at -80 °C for few days. However, this might lead to a certain level of degradation and EVs are thus better to be used on the day of preparation. EVs can also be filtered through 0.22 µm membrane microfilters to ensure their sterility, and to break down EV aggregates, before using them for any application.
  15. After P70K ultracentrifugation, proceed as previously for P35K EVs, transfer the supernatant in new ultracentrifugation tubes and subject these tubes to a 100,000 x g ultracentrifugation (100K) at 4 °C for at least 1 h (the longer, the higher yield of exosomes will be reached) (Figure 6).


    Figure 6. Non-diluted and PBS-diluted milk led to the formation of casein jelly after 70,000 x g ultracentrifugation. Dilution with sodium citrate leads to small EV-rich pellet with very little casein contamination. A. 70,000 x g pellet from non-treated milk. B. 70,000 x g pellet from diluted milk 1:1 in PBS. C. 70,000 x g pellet from diluted milk 1:1 in 2% sodium citrate.
    Note: Discard 70K pellet or keep it if interested by its content (Figure 6C). If so, proceed to its suspension as for P35K EVs.

  16. After P100K ultracentrifugation, transfer the 100K supernatant to new ultracentrifugation tubes and subject to 100,000 x g ultracentrifugation for 18 h at 4 °C if you wish to obtain an EV-free supernatant to use as a control.
  17. Suspend 100K pellet (Figure 7C) the same way as for previous pellets and store EVs the same way.


    Figure 7. Non-diluted and PBS-diluted milk led to the formation of casein jelly after 100,000 x g ultracentrifugation. Dilution with sodium citrate leads to small EV-rich pellet with very little casein contamination. A. 100,000 x g pellet from non-treated milk. B. 100,000 x g pellet from diluted milk 1:1 in PBS. C. 100,000 x g pellet from diluted milk 1:1 in 2% sodium citrate.

Data analysis

This protocol does not generate data but functional EVs. The quality and content of these can be assessed through different approaches.

  1. EV concentration
    EVs content of the final pellets can be indirectly quantified by assessing their protein content using Bradford's reagent (Sigma-Aldrich) or BCA Protein Assay Kit (Millipore), by isolating total RNA and determining its concentration (NanoDrop, Bioanalyzer, Qbit, etc.) or more precisely by directly quantifying EVs using high sensitivity flow cytometry (Benmoussa et al., 2017; Morales-Kastresana and Jones, 2017) or nanoparticle tracking analysis (NTA) (Gardiner et al., 2013).
  2. EV characteristics and quality
    Cow’s milk EVs characteristics can be routinely checked using densitometry, dynamic light scattering (DLS) and electron microscopy as previously described (Benmoussa et al., 2017).
  3. EV characterization
    We provided in our previous publications methodologies to analyze milk EV’s proteins (Benmoussa et al., 2019), small RNAs (Benmoussa et al., 2019c), resistance to digestion and bioaccessibility (Benmoussa et al., 2016) and bioactivity (Benmoussa et al., 2019a). These methodologies include EV density analysis, exploration of protein content by western blot, proteins profile by liquid-chromatography tandem mass spectrometry, small RNA and mRNA profiling by RNA microarray or next generation sequencing, TIM-1 in vitro digestion, dual luciferase assays, etc.
  4. EV functional activity
    Depending on the expected outcomes, different approaches might be suitable, and we would recommend to anyone exploring the field of milk EVs to read and follow MISEV2018 guidelines (Thery et al., 2018). For those exploring milk EVs biological activity on inflammation when delivered orally, we would recommend exploring, along with MISEV2018, the report associated to this protocol (Benmoussa et al., 2019a).

Notes

Through the setup of this protocol, we discovered that addition of sodium citrate (used at a final concentration of 1%) made cow’s milk translucid, comparable to plasma in appearance and viscosity. More importantly, it prevented the co-isolation of milk caseins and other milk proteins with different milk EV subsets (Famelart et al., 1998; Kort, 2012; Benmoussa et al., 2017), which confirmed the importance of calcium in casein gel formation upon ultracentrifugation. It is likely that our discovery of different EV subset in cow’s milk was made possible by the change in milk viscosity induced by sodium citrate (Benmoussa et al., 2017). This change in viscosity would also explain the relative purity of the milk EVs we isolated in our previous work, as it may prevent sedimentation of certain proteins or aggregates at the considered speeds (Benmoussa et al., 2016, 2017 and 2019b).
  Importantly, we diluted milk in a 1:1 ratio with a 2% sodium citrate solution, rather than dissolving citrate crystals in milk, which would have changed the biological properties (e.g., viscosity) of milk even more and possibly impeding the isolation of certain EVs (Momen-Heravi et al., 2012).
  Also, in our hands, EDTA used at a concentration sufficient to chelate all calcium in milk, did not have the same effect on milk as sodium citrate, and actually causes casein precipitation (Wolf et al., 2015). Also, in our previous works, we found that acidified milk or preprocessing with EDTA led to changes in microRNA content in EVs and exosomes in comparison to whole milk (Benmoussa et al., 2019c). EDTA may differ from the other chelators discussed above in that it may not provide the sodium that links to caseins to form soluble sodium caseinates (de Kort et al., 2009, 2011 and 2012; Kort et al., 2012).
  This protocol has shown robust reproducibility over 5 years of use within the hands of more than 7 manipulators. It worked with different bovine cow milks (raw and commercial pasteurized milk) and would be believed to function with milk from different species.
  It is, however, of importance to note that differential ultracentrifugation is a long process that, in certain conditions, can have deleterious effects on EVs quality (Thery et al., 2018). Filtration steps might also discard some EVs of importance. Therefore, one should always characterize the full content of milk EVs before discarding potentially important populations and compare the effects of the EVs before and after filtering. It might also be important to consider other approaches to isolate milk EVs and for which sodium citrate is compatible. As it is known to break casein micelles, which are roughly the size of EVs (~200 nm), sodium citrate might provide the means to isolate milk EVs through tangential filtration or continuous diafiltration (Phan et al., 2014; Busatto et al., 2018), and help with the use of size-exclusion chromatography, by relieving the need to discard caseins (Blans et al., 2017). It may even be considered for isolating EVs from industrial scale volumes of milk, in a continuous way, using simulated moving bed size-exclusion chromatography (Satzer et al., 2014). It is also important to note that the use of sodium citrate might not be compatible with other approaches or milks from other species. Validation should be done prior to any long-term / large-scale implementation of this methodology.
  In any case, and independently of the chosen protocol, filling the 2018 MISEV guideline checklist and attaching it to the chosen protocol is highly recommended to ensure replicability and proper reporting in milk EVs studies.

Recipes

  1. 2% sodium citrate solution (1 L)
    1. Dilute 20 g of sodium citrate dihydrate in 1 L of autoclaved water filtered through 0.22 µm membrane microfilters or in autoclaved Milli-Q water
    2. Stir for 10 min using magnetic stirrer
    3. Filter through 0.22 µm membrane microfilters
    Note: You can also prepare a stock solution at 10% sodium citrate and dilute it 1/10th upon need. The solution can be stored at room temperature for few weeks. There is no need for pH adjustment.
  2. Phosphate buffer saline (PBS) 10x (1 L)
    In 800 ml of Milli-Q water
    1. Add 8 g of NaCl (preferably low in endotoxins, e.g., Millipore-Sigma, SAFC)
    2. Add 0.2 g of KCl
    3. Add 1.44 g of Na2HPO4
    4. Add 0.24 g of KH2PO4
    5. Adjust the pH to 7.4 with HCl
    6. Complete with Milli-Q water to a total volume of 1 L
    7. Filter the solution through 0.22 µm membrane microfilters
      Note: Solution can be autoclaved to ensure further sterility.
    8. Dilute 10x stock solution to 1x and before using it, filter the diluted PBS through 0.22 µm membrane microfilters
      Note: Stock solution can be stored at room temperature. Diluted solutions should be stored in sterile conditions at 4 °C to avoid contamination for few weeks.
  3. EDTA 0.5 M recipe (100 ml)
    In 80 ml of Milli-Q water:
    1. Add 18.6 g of EDTA
    2. Stir using magnetic stirrer for 10 min
    3. Adjust pH to 8.0 using NaOH in Milli-Q water solution
    4. Adjust to 100 ml
    5. Complete with Milli-Q water to a total volume of 100 ml and filter it through 0.22 µm membrane microfilters
    Note: The solution can be autoclaved to ensure further sterility and stored at room temperature for few weeks.

Acknowledgments

This work was supported by the Canadian Institutes of Health Research (CIHR) Grants No. IG1-134171, MOP-137081 (through the Institute of Genetics) and PJT-165806 (to P.P.). A.B. (No. 262093) received a PhD studentship award from the FRQ-S. Authors wish to acknowledge Patricia Savard for her help in solving casein jelly formation. This protocol was previously used in our reported works (Benmoussa et al., 2016, 2017, 2019a, 2019b and 2019c).

Competing interests

The author(s) declare no competing interests and, as we worked with commercially available cow milk, the authors were under no requirement of ethical committee approval.

References

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  14. Kort, E.J.P. de. (2012). Influence of calcium chelators on concentrated micellar casein solutions: from micellar structure to viscosity and heat stability. In: Kort, E. J. P. de. (Ed). ISBN: 978-94-6173-237-8.
  15. de Kort, E., Minor, M., Snoeren, T., Hooijdonk, T. and Linden, E. (2011). Effect of calcium chelators on physical changes in casein micelles in concentrated micellar casein solutions. Int Dairy J 21(12): 907-913.
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简介

[摘要 ] 牛奶是一种复杂的流体,其中包含各种类型的蛋白质和细胞外囊泡(EVs),有些蛋白质会与EV混合在一起,并干扰其分离,在这些蛋白质中,酪蛋白形成的胶束大小与牛奶EV相当因此,可以将其与电动汽车共隔离。影响牛奶的初步步骤对于电动汽车的隔离,影响分离电动汽车的纯度和丰度至关重要。在我们以前对牛奶电动汽车的研究过程中,我们发现柠檬酸钠(最终含量为1% )是一种生物相容性试剂,能够将酪蛋白胶束分解为40 nm单体,可分离出大量的EV,而酪蛋白或其他污染蛋白的沉淀率却很低。 EV子集,深入表征其形态,蛋白质含量和小分子RNA富集模式。我们还能够描述其在小鼠肠道炎症模型中的生物学功能。具体来说,是从同一样品中分离出不同乳EV子集的培养基。更具体地说,我们着重介绍了使用柠檬酸钠作为标准化方法来分离和研究乳EV的方法及其在差分超速离心之外的分离技术的潜力。

[背景 ] 在我们以前的出版物(本穆萨等人。,2016 ,2017,2019b和2019c;本穆萨和普罗沃斯特,2019) ,我们强调,牛乳是包含细胞外无数的复杂流体囊泡。(EV)用的子集在这些之中,外泌体是多囊体(MVB)与细胞膜融合时释放的约100 nm囊泡,进行超速离心时,这些沉淀物的离心速度等于或高于100,000 xg (P100K,其中P代表沉淀)(Pieters 等。,2015) 。其他非外来体EV子集在牛奶以较低的速度沉积物(发现和媲美外来体,在形状和尺寸,但例如,12000 ×g离心,P35K ; 35000 ×g离心,P35K ; 70000 ×g离心,P100K)这些是你GHT是源自细胞膜的出芽和容纳从外来体内容不同的特定蛋白质和微RNA (本穆萨等人。,2016 ,2017 ,2019b和2019c;本穆萨和普罗沃斯特,2019) 。

长期以来,由于它们的形态和化学相似性,分离这些不同的乳EV子集仍然具有挑战性。更多,乳EV研究中使用的规程通常集中在乳外泌体的分离上,从而导致生物活性非外泌乳电动车(Benmoussa 等人,2019a,2019b和2019c)。

当使用差异超速离心法分离牛奶EV时,第一步通常会因牛奶蛋白与EV的共沉淀而受损,这会在试管底部形成致密的果冻(Zonneveld et al。,2014)。当酪蛋白受到较高的机械压力时(Famelart 等,1998; Zonneveld 等,2014)。由于这种果冻非常稠密,几乎无法重悬,因此尚不清楚是否某些EV亚型被困在其基质中和一些协议建议丢弃这种富含酪蛋白的果冻。

某些报告建议使用密度梯度或缓冲垫来防止电动汽车与酪蛋白果冻混合(Zonneveld 等人,2014)。其他报告则建议,在对牛奶“血清”或乳清进行差异化处理之前,应先丢弃酪蛋白来避免这种果冻的形成。 ultracentrifugation.To为此,牛奶通常与酸混合(Somiya 等人,2018)或用冷的EDTA以沉淀之前奶电动汽车沉淀酪蛋白(沃尔夫等人,2015) 。然而,生物体液的这样的预处理有这对细胞外囊泡(EV)的分离,质量和产量产生了巨大影响(Zonneveld et al。,2014),还需要丢弃低速弹丸的EV(P12K或P35K)。此外,缺乏信息关于酸化或酪蛋白沉淀对牛奶电动车的影响,以及在此过程中某些电动车可能丢失的可能性。

这一点尤其重要,因为牛奶中有多个EV子集(Benmoussa 等人,2017和2019b),而且乳清与牛奶相比具有不同的microRNA和蛋白质含量(Benmoussa 等人,2019b; Benmoussa和Provost,2019),其中,因为微RNA与发现在电动汽车,表明情况precipitation.This在某些电动汽车的损失也通过在乳来源富含酪蛋白的产品,像奶酪微RNA的发现支持(本穆萨和普罗沃斯特,2019)。还有,牛奶电动车的生物活性以及它们运输的生物活性分子在很大程度上取决于该液体所暴露或经受的试剂/预处理步骤和分离规程(Zonneveld et al。,2014)。

遇到此问题时,我们在不同的地方进行了实验,以免沉淀酪蛋白奶,同时确保分离出不同结构保守和功能性的EV乳子集。在牛奶中,酪蛋白以70-200 nm的胶束形式排列直径,接近某些电动汽车子集的大小(Blans 等人,2017)。了解钙对于维持这些胶束很重要(de Kort 等人,2009、2011 和2012; Kort 等人,2012),我们认为钙螯合会阻止酪蛋白上层结构的形成,从而阻碍超速离心分离电动汽车。

先前已经研究了几种钙螯合剂破坏酪蛋白胶束的能力,包括尿苷一磷酸二钠(Na 2 UMP),磷酸二钠(Na 2 HPO 4 ),柠檬酸三钠(简称为“柠檬酸钠”),钠。植酸盐,六偏磷酸钠(SHMP)或乙二胺四乙酸(EDTA)(Ward 等人,1997; de Kort 等人,2009,2011和2012; Kort 等人,2012)。这些螯合剂与胶体磷酸钙相互作用( CCP)导致酪蛋白胶束分解成小的色素。该过程增强了牛奶在加热过程中的稳定性,并改变了牛奶的物理性能,通常会降低其粘度(de Kort 等人,2009、2011和2012; Kort 等人, 2012)。

其中,柠檬酸钠是生物相容的化合物已知的1 00 Tasu 年,它有能力以防止酪蛋白凝结在胃的婴儿(Poynton,1904) ,它被广泛用于保护生物流体,如血(Janse范·伦斯堡和Van der Merwe,2017)作为食品添加剂和世界卫生组织推荐的主要补液盐形式(Pizarro 等,1986; Banipal 等,2016)。值得注意的是,它以前曾被用来破坏酪蛋白胶束。有助于通过渗滤分离乳脂球(MFG)并防止孔堵塞(Phan et al。,2014)。有趣的是,柠檬酸钠在高机械压力下会特别影响乳蛋白凝胶的形成,尽管其潜在机制尚不清楚,除了它对溶液pH值的依赖性(Famelart 等,1998)。

在柠檬酸钠的实验过程中,我们发现用柠檬酸钠(最终含量为1%)对牛奶进行预处理是一种经济有效的方法,可以避免凝胶化和使用差速超速离心分离大量不同牛奶EV的情况。详细介绍了该方法的基础方法,这些方法导致了牛奶中不同电动汽车子集的发现(Benmoussa 等人,2016和2017)和表征(Benmoussa 等人,2017、2019b和2019c),使其保持功能并能够转移其共ntent到人类细胞(本穆萨等人,2019c).EVs,包括外来体,通过这种方法分离的能够实验性结肠炎中,以调节肠道炎症(本穆萨等人,2019a) 。

关键字:细胞外基质, Evs, 牛乳, 外泌体, 细胞微泡, 超速离心, 牛乳, 酪蛋白, 柠檬酸盐

材料和试剂


 


消毒玻璃瓶(派热克斯,康宁生命科学公司,目录号:1395-1L)或无菌塑料瓶,用于混合牛奶和柠檬酸钠(康宁,目录号:431533)
管50毫升猎鹰(任何产品,所述只要在无菌和无热原的,例如,康宁,目录数目:352070,费舍尔小号系统求解,目录号的:14-432-22)
1.7 ml卡口盖管(任何无菌且无热原的产品,例如,Corning,Costar via Sigma - Aldrich,目录号:CLS3620)
0.22 µm膜微过滤器(Corning,Sigma-Aldrich,目录号:CLS431224)
血清学吸管不同体积(只要无菌的和无热原的,任何产品例如,Sigma-Aldrich公司,目录号:SIAL1485)
过滤1毫升枪头(任何产品只要无菌的,无热原和过滤,例如,热电科学,Ç atalog Ñ 棕土:94052410)
丁腈一次性手套(任何类型)
无菌湿巾/纱布(只要无菌单包装,例如,VWR,目录号:CA95041-740)
可选:EDTA 。可以作为即用型溶液(Sigma-Millipore,目录号:324506)购买,也可以从EDTA粉中自制
牛奶
在实验当天使用市售的牛奶或在“无菌条件”下收集和储存的原牛奶(使用无菌瓶尽可能无菌并保持瓶盖封闭直至进入生物罩)。多数为巴氏杀菌的商业超滤脱脂牛奶(Lactantia PureFilter ,在当地杂货店购买),因为它避免了可能导致差异的乳化步骤。我们建议使用三杯具有不同抢先日期的牛奶,牛奶必须在4 °C下储存。


注意:如果使用生奶,请使用不同奶牛的奶池以最大程度地减少个体差异。非商业生或巴氏灭菌的新鲜奶可以在-80 °C 下冷冻以避免细菌污染/扩散,但请注意,这样的储存可能会影响电动汽车的容量。牛奶,请确保事先进行脱脂和脱细胞处理,以最大程度减少因乳脂球或冷冻/融化循环而破坏细胞而引起的电动汽车形成。以小等份(50毫升)的速度冷冻牛奶,这样在融化时它会更快地达到熔点并限制电动汽车破坏。


布拉德福德氏试剂(Sigma-Aldrich,目录号:B6916)
BCA蛋白测定试剂盒(Millipore,目录号:71285-M)
Milli-Q水或任何过滤的超纯水(例如,Millipore,水系统清晰分选和过滤,目录号:ZRXQ003WW)
的NaCl(p 中内毒素referably低,例如,Millipore公司的西格玛,SAFC,目录号:1.16224)
KCl(例如Sigma-Aldrich,目录号:P3911)
Na 2 HPO 4 (例如Sigma-Aldrich,目录号:NIST2186II)
KH 2 PO 4 (例如Sigma-Aldrich,目录号:NIST200B)
HCl(例如Sigma-Aldrich,目录号:320331)
EDTA(例如Sigma-Aldrich,目录号:EDS-500G)
Milli-Q水中的NaOH溶液(例如Sigma-Aldrich,目录号:S8045)
柠檬酸钠二水合物(Sigma-Aldrich,目录号:W302600)
磷酸盐缓冲溶液
可以购买市售的pH 7.4无菌磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P5493)或自制。


2%柠檬酸钠溶液(1 L)(请参阅食谱)
磷酸盐缓冲盐水(PBS)10x(1 L)(请参阅食谱)
EDTA 0.5 M配方(100 ml)(请参阅食谱)
 


设备


 


与转子波纹管兼容的超速离心管(36 ml,Beckman,目录号:355631或344058或其他,请参见转子兼容性:https ://assets.thermofisher.com/TFS-Assets/LED/Specification-Sheets/Thermo-Scientific- SureSpin-630-(36 ml)-转子.pdf)
超速离心机SORVALL WX超速离心机(或一种热科学TM SORVALL TM WX 超速离心机,产品目录号:75000100,7500090,7500080)
转子SureSpin 630摆动铲斗转子(Thermo Scientific,目录号:79368)
注意:您可以使用其他离心机和其他转子,但要确保使用可比的材料。请注意,不建议使用定角转子,因为它们会导致更高的EV退化。


移液器(任何类型,例如通过Mandel的Drummond Scientific便携式Pipet-Aid XP,目录号:DRU-4-000-101)
轨道摇床(任何类型,例如,Bel- 艺术TM SP Scienceware TM Spindrive TM 轨道摇床平台,经由费舍尔科学,目录号:1451176)
旋转混合器/管式左轮手枪(任何类型,例如,热科学型,目录号:88881001)
高精密天平(任何类型,例如,赛多利斯,Ç atalog号:PRACTUM224-1S)
试管架不同尺寸(任何产品)
大号aminar流罩(如果工作在无菌条件)(任何产品)
微量,不同体积(任何类型的,只要它们是精确和移液是足够平滑,以避免液体悬浮液,过快喷出例如,移液器大号P1000L,100- 1000微升,金属喷射器,吉尔森,Ç atalog号:FA10006M)
-80°C冷冻室
磁力搅拌器
 


程序


 


注意:所有步骤均应在4 °C下进行。


开始超速离心并将装置温度设置为4 °C。
在无菌玻璃瓶或塑料瓶中混合125毫升脱脂牛奶和2%柠檬酸钠1:1。
注意:先倒入柠檬酸钠,然后倒入牛奶以加速酪蛋白胶束的破裂。


保持在摇15分钟,在4 ℃下。确保牛奶是轻轻mixi网纳克(1/4 个最大速度)。
注意:这可以在冷藏室中完成,也可以在摇杆上放冰,在冰上放牛奶。


15分钟后,应澄清牛奶(使其具有与血浆相当的半透明状态),并准备装入超速离心管中(图1)。
 


D:\ Reformatting \ 2020-3-2 \ 1903005--1382 Patrick Provost 837236 \ Figs jpg \图1.jpg


图1.将脱脂牛奶与2%柠檬酸钠1:1混合后,在摇杆上于4 °C 孵育15分钟后,澄清牛奶


笔记:


添加碳酸钙会使澄清相反,从而证实该过程中钙螯合的参与。
如果您使用的牛奶没有澄清,您可以将柠檬酸钠的浓度增加到3 %或4%。
 


用牛奶-柠檬酸钠混合物完全充满超透明离心管(图2)。
注:制作确保管被完全填充或者他们可能在离心打破。


将管放入转子管支架中,并使用无菌移液管通过在相对管之间转移牛奶-柠檬酸钠混合物来保持相对管之间的质量(图2)(保持管,柠檬奶溶液,管支架和盖的可比添加质量) )。
 


D:\ Reformatting \ 2020-3-2 \ 1903005--1382 Patrick Provost 837236 \ Figs jpg \图2.jpg


图2. Ultraclear 厚超速离心管应该被加载到管保持器的质量平衡为超速离心前


 


盖上盖子,将它们放在要使用的插座中(由管座和转子上的数字指示,图3)。
 


D:\ Reformatting \ 2020-3-2 \ 1903005--1382 Patrick Provost 837236 \ Figs jpg \图3.jpg


图3.确保将SureSpin 管装入正确的插座中,以避免平衡问题


 


旋转电动汽车是理想的速度。在我们的协议中,我们在4 °C下以35,000 xg (35K)的速度分离了2小时的低速沉降电动汽车。我们使用了Sorvall WX TL-100超速离心机的K因子自动计算并将加速度设置为A = 9并在D = 9时中断。
注意:如果使用另一台超速离心机,请确保考虑到转子角度和k因子将g力的速度转换为适当的每分钟转数(RPM)。如果希望使用不同的速度,请参考转子和离心机制造商的建议(https ://Assets.Thermofisher.Com/TFS-Assets/LED/Specification-Sheets/Thermo-Scientific-SureSpin-630-(36 Ml)-Rotor.Pdf。)如果使用生乳,则可以使用脱牛奶使液体脱细胞在4 °C下以1,000 xg离心10分钟,然后以4,500 xg离心30分钟,然后进行上述操作。


2小时后,电动汽车应已沉淀,在试管底部形成半透明的凝胶状沉淀(图4 C )。
 


D:\ Reformatting \ 2020-3-2 \ 1903005--1382 Patrick Provost 837236 \ Figs jpg \图4.jpg


4.图35,000 Xg 超速离心后导致酪蛋白冻形成的未稀释和PBS稀释的牛奶。用柠檬酸钠稀释会导致EV含量高的小颗粒,酪蛋白污染极小。A. 从未处理的牛奶中分离出35,000 Xg的颗粒。B. 35000 X ģ 颗粒从稀奶1:1在PBS中。C. 35000 XG 颗粒从稀奶1:1在2 Pasento柠檬酸钠含有非常少的污染性酪蛋白。


注意:右边的小球包含P35K电动车,在丢弃印迹和悬浮液之前可能看不到,它固定在试管底部,因此在接下来的步骤中丢失的风险很小。


 


将克隆转移到新的干净/无菌超速离心管中。
注意:这可以使用血清移液器和移液器完成,也可以直接将上清液倒入新的试管中,具体取决于所需的无菌水平。


平衡上清液试管,并按照与前面相同的步骤在4 °C下以70,00 0 xg (70K)离心1 h 。
在70K超速离心过程中,将试管架上剩余的无液的含35K颗粒的试管放回,试管的底部带有无菌纱布/纸,试管的开口接触纱布/纸以使残留的液体被丢弃。纱布/纸内的毛细作用(图5)。
 


D:\ Reformatting \ 2020-3-2 \ 1903005--1382 Patrick Provost 837236 \ Figs jpg \图5.jpg


图5. 纸管上的翻转管允许残留的液体因电容而被丢弃


注意:这是重要的,以避免污染与来自蛋白质印迹,并保持精确的悬浮液volumes.You可以使用移液管和提示,以丢弃在管内的任何剩余的液体或无菌纱布/纸。


 


将所有沉淀(6管)悬浮在相同的1 ml无菌过滤PBS溶液中。
笔记:


避免用尖头弄碎。使用移液器和1毫升过滤的尖头将PBS滴在其上,然后用PBS轻轻地从侧面开始“侵蚀”沉淀物,然后轻轻地到达中心。尝试快速有效地避免颗粒干燥,但避免粗略混合,否则最终会出现泡沫受限的颗粒悬浮液。
你也可以倒100 μ升PBS在管上,同时使用500您没有悬浮μ 升PBS暂停第一管。转移500 μ 升具有从而600第二管μ 升用于第二沉淀,等on.Depend你的应用,如果计划进行qPCR或进行功能研究,则可以在PBS中加入0.5%EDTA,以谨慎地进行悬浮。
将悬浮的P35K电动汽车转移到1.7 ml Eppendorf管中,并在4 °C 的旋转混合器中至少放置24 h,以使P35K电动汽车完全悬浮,然后将其在-80 °C 下保存几天。可能会导致一定程度的降解,因此在制备当天最好使用电动汽车。电动汽车还可以通过0.22 µm膜微滤器过滤,以确保其无菌性,并分解电动汽车聚集体,然后将其用于任何应用。
在进行P70K超速离心后,对P35K电动汽车进行以前的处理,将稳定剂转移到新的超速离心管中,并在4 °C的条件下对这些管进行100,000 xg 超离心(100K)至少1 h(时间越长,外泌体的产量就越高)达到)(图6)。
 


D:\ Reformatting \ 2020-3-2 \ 1903005--1382 Patrick Provost 837236 \ Figs jpg \图6.jpg


图6 。非稀释,PBS,稀释牛奶导致后70,000酪果冻的形成XG 超速离心。稀释柠檬酸钠导致小型EV-丰富的颗粒非常小酪蛋白污染。A. 70000 XG 颗粒从未经处理的牛奶。B. 70000 XG 颗粒从稀奶1:1.在PBS C. 70000 XG 颗粒从稀奶1:1在2 Pasento小号裂果柠檬酸盐。


注意:丢弃70K的小球或对其内容感兴趣的话将其保留(图6 C)。如果是这样,请像P35K电动汽车一样继续将其暂停。


 


P100K超速离心后,如果希望获得无EV的昆虫作为对照,请将100K电容器转移到新的超速离心管中,并在4 °C下进行100,000 x g 超速离心18 h 。
与以前的颗粒相同,将100K颗粒悬浮(图7 C),并以相同的方式存储EV。
 


D:\ Reformatting \ 2020-3-2 \ 1903005--1382 Patrick Provost 837236 \ Figs jpg \图7.jpg


图7 。非稀释,PBS,稀释牛奶导致酪果冻后形成10万XG 超速离心。稀释柠檬酸钠导致小型EV-丰富的颗粒非常小酪蛋白污染。A. 100000 XG 颗粒从未经处理的牛奶。B. 100000 XG 颗粒从稀奶1:1.在PBS C. 100000 XG 颗粒从稀奶1:1在2 Pasento柠檬酸钠。


 


数据一nalysis


 


该协议不会生成数据,但会生成功能电动汽车,这些电动汽车的质量和内容可以通过不同的方法进行评估。


电动汽车浓度
可通过使用Bradford's试剂(Sigma-Aldrich)或BCA蛋白测定试剂盒(Millipore)评估其蛋白质含量,通过分离总RNA并确定其浓度(Nano D Rop ,生物分析仪,Qbit,Etc等)来间接定量最终颗粒的EVs含量。)或更精确地,通过使用高灵敏度的流式细胞术直接定量电动汽车(本穆萨等人,2017; Morales的-Kastresana和Jones,2017)或纳米粒子追踪分析(NTA) (加德纳。等人,2013年)。


电动汽车的特性和质量
如前所述(Benmoussa et al。,2017),可以使用光度法,动态光散射(DLS)和电子显微镜常规检查牛奶EVs特性。


电动汽车表征
我们在以前的出版物中提供了方法来分析牛奶EV的蛋白质(Benmoussa 等人,2019),小RNA (Benmoussa 等人,2019c),抗消化性和生物可及性(Benmoussa 等人,2016)以及生物活性(Benmoussa 等人,2016)。等,2019a)。这些方法包括EV密度分析,通过蛋白质印迹探索蛋白质含量,通过液相色谱串联质谱法进行蛋白质谱分析,通过RNA芯片或下一代测序进行小RNA和mRNA分析,TIM-1 体外消化。 ,双重萤光素酶测定法等。


EV功能活动
根据预期的结果,可能会采用不同的方法,我们会建议任何探索牛奶电动车领域的人阅读并遵循MISEV2018指南(Thery 等人,2018)。口头上,我们建议与MISEV2018一起探索与该协议相关的报告(Benmoussa et al。,2019a)。


 


笔记


 


                                                        通过该协议的设置,我们发现添加柠檬酸钠(以1%的最终浓度使用)可使牛乳透明,在外观和粘度方面可与血浆媲美,更重要的是,它防止了酪蛋白和牛奶的共分离。其他具有不同乳EV子集的乳蛋白(Famelart 等,1998; Kort,2012; Benmoussa 等,2017),这证实了钙在超速离心酪蛋白凝胶形成中的重要性。我们发现不同的EV柠檬酸钠引起的牛奶粘度变化使得牛奶中的一部分成为可能(Benmoussa et al。,2017)。这种粘度变化也可以解释我们在先前工作中分离出的牛奶电动汽车的相对纯度,因为它可能在所考虑的速度某些蛋白质或聚集的防止沉淀(本穆萨等人。,2016 ,2017 和2019b) 。


  重要的是,我们用2%的柠檬酸钠溶液以1:1的比例稀释了牛奶,而不是将柠檬酸盐晶体溶解在牛奶中,这将更大程度地改变牛奶的生物学特性(例如粘度),并可能阻碍某些特定食品的分离。电动汽车(Momen-Heravi et al。,2012)。


  此外,在我们的手中,EDTA使用浓度足以螯合全部的钙在牛奶,并没有同样的效果在牛奶为柠檬酸钠,并且实际上导致酪蛋白沉淀(沃尔夫等人,2015年)。此外,我们在以前的作品,我们发现,酸化的乳或用EDTA预处理导致在电动汽车和外来体微小RNA含量的变化相比于全脂奶(本穆萨等人,2019c).EDTA 可以从上面的,因为它可能不提供所讨论的其它螯合剂不同与酪蛋白连接形成可溶性酪蛋白酸钠的钠(de Kort 等,2009,2011 和2012; Kort 等,2012)。    


  该协议已在超过7个操纵器的手中使用了5年,显示出强大的可重复性,可与不同的牛乳(原料奶和市售巴氏杀菌奶)一起使用,并被认为可与不同种类的牛奶一起使用。


  然而,值得注意的是,差异超速离心是一个漫长的过程,在某些条件下可能会对电动汽车的质量产生有害影响(Thery 等人,2018)。过滤步骤也可能会丢弃一些重要的电动汽车。每个人都应该表征全部内容牛奶电动车丢弃之前了Ing 潜在的重要群体,并比较电动车前和过滤后的效果。这也可能是重要的考虑其他途径来隔离乳电动车和哪个柠檬酸钠是兼容的。作为已知会破坏酪蛋白微胶束,胶束的大小大约是电动车(〜200 nm)的大小,柠檬酸钠可能提供了通过切向过滤或连续渗滤来分离牛奶电动车的方法(Phan 等人,2014; Busatto 等人, (2018),并通过消除排泄酪蛋白的需求来帮助使用体积排阻色谱法(Blans 等,2017)。甚至可以考虑从工业规模的体积中分离电动汽车 使用模拟移动床尺寸-排阻色谱法以连续方式连续提取Es牛奶(Satzer Et Al。,2014)。还需要注意的是,柠檬酸钠的使用可能与其他方法或其他种类的牛奶不兼容验证应在此方法的任何长期/大规模实施之前进行。


  在任何情况下,无论选择哪种方案,都强烈建议填写2018 MISEV指南清单并将其附加到所选方案上,以确保在牛奶电动汽车研究中具有可重复性和适当的报告(https://cdn.ymaws.com/www。 isev.org/resource/resmgr/misev2018_checklist.pdf)。


 


菜谱


 


2%柠檬酸钠溶液(1 L)
在通过0.22 µm膜微滤器过滤的1 L高压灭菌水中或高压灭菌的Milli-Q水中稀释20 g柠檬酸钠二水合物
使用磁力搅拌器搅拌10分钟
通过0.22 µm膜微滤器过滤
注意:您也可以准备10%柠檬酸钠的原液并根据需要稀释1/10,该溶液可在室温下保存数周,无需调节pH。


磷酸盐缓冲盐水(PBS)10x(1 L)
在800毫升Milli-Q水中


加入8克NaCl(p 中内毒素referably低,例如,Millipore公司-Σ,SAFC)
加入0.2克KCl
加入1.44克Na 2 HPO 4
加入0.24 g KH 2 PO 4
用HCl将pH调节至7.4
配以Milli-Q水,总体积为1 L
通过0.22 µm膜微滤器过滤溶液
注意:溶液可以高压灭菌以确保进一步无菌。


将10倍原液稀释至1倍,然后使用,将稀释的PBS通过0.22 µm膜微滤器过滤
注意:原液可在室温下保存,稀释后的溶液应在4 °C的无菌条件下保存,以免污染数周。


EDTA 0.5 M配方(100毫升)
                                          在80毫升Milli-Q水中:


加入18.6克EDTA
使用磁力搅拌器搅拌10分钟
使用Milli-Q水溶液中的NaOH将pH调节至8.0
调整至100毫升
装满Milli-Q水至总体积为100 ml,并通过0.22 µm膜微滤器过滤
注意:溶液可以高压灭菌以确保进一步无菌,并在室温下保存数周。


 


致谢


 


这项工作得到了加拿大卫生研究所(CIHR)的资助,编号IG1-134171,MOP-137081(通过遗传学研究所)和PJT-165806(授予PP).AB(编号262093)获得了博士学位作者希望感谢Patricia Savard在解决酪蛋白果冻形成方面的帮助,该协议以前曾在我们报道的作品中使用(Benmoussa 等人,2016、2017、2019a,2019b和2019c)。


 


利益争夺


 


作者声明没有利益冲突,并且,由于我们使用市售牛奶进行工作,因此作者无需获得伦理委员会的批准。


 


参考文献


 


Banipal,TS,Kaur,H.,Kaur,A.和Banipal,PK(2016)。基于酒石酸盐和柠檬酸盐的食品添加剂对十二烷基硫酸钠的胶束性质的影响,有望用作食品乳化剂。食品化学190:599-606 。              
Benmoussa,A.,Diallo,I.,Salem,M.,Michel,S.,Gilbert,C.,Sevigny,J.和Provost,P.(2019a)。牛奶中细胞外囊泡的两个子集的浓缩物可调节症状。和炎症在试验colitis.Sci代表9(1):14661。              
Benmoussa,A.,Gotti,C.,Bourassa,S.,Gilbert,C.和Provost,P.(2019b)。牛奶中细胞外囊泡(EV)子集的蛋白质标记物的鉴定 .J Proteomics 192:78-88。 。              
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引用:Benmoussa, A., Michel, S., Gilbert, C. and Provost, P. (2020). Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation. Bio-protocol 10(11): e3636. DOI: 10.21769/BioProtoc.3636.
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