Isolation, Purification and Characterization of Exosomes from Fibroblast Cultures of Skeletal Muscle
骨骼肌成纤维细胞培养物中外泌体的分离、纯化、鉴定
发布: 2020年04月05日第10卷第7期 DOI: 10.21769/BioProtoc.3576 浏览次数: 7767
评审: Alka MehraKristina Aguilerailgen Mender
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参见作者原研究论文
The authors used this protocol in:
Jul 2019
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Abstract
Exosomes are dynamic nanovesicles secreted by virtually all cells and are present in all biological fluids. Given their highly heterogeneous content exosomes have been implicated in many physiological and pathological processes that they exert by influencing cell-cell and cell-ECM communication. In recent years an increasing number of methods have been established for the purification and characterization of exosomes. These include ultracentrifugation, ultrafiltration, size exclusion chromatography, immune capture and precipitation using a proprietary polymer. Here, we provide a protocol based on differential ultracentrifugation and sucrose density gradients tailored for the isolation of crude and ultra-pure exosomes from primary fibroblast cultures derived from adult mouse skeletal muscle. This protocol can be adapted and modified for the isolation and characterization of exosomes from a variety of tissues and bodily fluids.
Keywords: Fibroblasts (成纤维细胞)
Cell culture (细胞培养)
Exosomes (外泌体)
Ultracentrifugation (超速离心)
Sucrose gradient (蔗糖梯度)
Western blot (免疫印迹)
Background
Exosomes are single-membrane, heterogeneous nanovesicles ranging in diameter from 30 to 150 nm, secreted by all cells and present in virtually all body fluids. The spectrum of soluble and membrane macromolecules, mRNAs, microRNAs present in exosomes depends on the metabolic status as well as the developmental origin of the cell(s) secreting these nanovesicles. Owing to their cargo composition, exosomes can initiate signaling pathways in the receiving cells and have been involved in development, immunity and in the maintenance of normal tissue physiology. In disease conditions, like neurodegeneration, fibrosis and cancer, they have been shown to trigger and propagate pathological stimuli (Rackov et al., 2018; Gurunathan et al., 2019; van de Vlekkert et al., 2019). Here we describe a protocol for the purification of exosomes from fibroblast cultures established from adult mouse gastrocnemius (GA) muscle (van de Vlekkert et al., 2019). These purified exosomal preparations can be subjected to mass spectrometry. The advantage of this method is that is scalable and cost effective. Primary mouse fibroblasts are kept in culture for no more than 4 passages to preserve their primary status, and their conditioned medium is then processed by differential centrifugation to obtain crude exosomal preparations that are further purified by sucrose density gradients. This protocol has been successfully used to assess and compare the protein composition of exosomes released by normal mouse fibroblasts and by activated fibroblasts (myofibroblasts) cultured from the GA muscle of WT and Neu1–/– mice, respectively (van de Vlekkert et al., 2019). The Neu1–/– mouse is a model of the lysosomal storage disease sialidosis, caused by deficiency of the sialidase neuraminidase 1 (NEU1) (de Geest et al., 2002; d’Azzo et al., 2015). Mouse cells deficient for Neu1, including primary myofibroblasts, have excessive exocytosis of soluble lysosomal contents as well as increased release of exosomes (van de Vlekkert et al., 2019). Exosomes purified from Neu1–/– myofibroblasts were shown to be potent carriers of fibrotic signaling molecules and to be responsible for eliciting a pathogenic cascade, which ultimately led to muscle fibrosis and degeneration (van de Vlekkert et al., 2019).
Materials and Reagents
Note: All materials and reagents are kept at room temperature unless otherwise described. For the shelf life and storage temperature of reagents, we refer to the manufacturer’s instructions.
- Advantus T-pins (Fisher scientific, catalog number: S174301)
- Light duty paper wipes (Georgia Pacific, Brawny, catalog number: 29221)
- Falcon 50 ml conical centrifuge tubes (Corning 352070) (Fisher Scientific, catalog number: 14-432-22 )
- 10-cm and 15-cm dishes (Midwest Scientific, TPP, catalog numbers: TP93100 and TP93150 respectively)
- Countess cell counting chamber slides and Trypan Blue (Thermo Fisher Scientific, Invitrogen, catalog number: C10228 )
- Thin wall Polypropylene tubes (25 x 89 mm) (Beckman Coulter, catalog number: 326823 )
- Ultra-clear tubes (25 x 89 mm) (Beckman Coulter, catalog number: 344058 )
- Ultra-clear tube (14 x 89 mm) (Beckman Coulter, catalog number: 344059 )
- Premium microcentrifuge tubes (1.7 ml) (Fisher Scientific, Midwest Scientific, catalog number: AVSC1510)
- Multiple well plates (96-well) (Midwest Scientific, TPP, catalog number: TP92696 )
- Protected disposable scalpels (stainless steel blade) (Fisher Scientific, Aspen Surgical Bard-Parker, catalog number: 372610 )
- Immobilon-P Transfer membranes (Millipore, catalog number: IPVH00010 )
- Blotting paper (Fisher, Whatmann, catalog number: 09-301-404)
- Sterile cell strainers (Thermo Fisher Scientific, Fisherbrand, catalog number: 22-363-548 )
- Sterile pipet 5 ml, 10 ml and 25 ml (Falcon, catalog numbers: 357543 , 357551 , 357525 , respectively）
- AvantGuard low-binding barrier tips
10 μl universal-long (Midwest Scientific, catalog number: ASAVANBARTIPS AV10R-H)
20 μl universal (Midwest Scientific, catalog number: ASAVANBARTIPS AV20H)
200 μl universal (Midwest Scientific, catalog number: AV200-H )
1,250 μl universal (Midwest Scientific, catalog number: AV1250H) - Gel round tips (Midwest Scientific, catalog number: MPT1000)
- Adult mice
Notes:- Both male and female mice can be used for this protocol.
- The gastrocnemius muscle is used as example in this protocol. However, the procedure is feasible with any other type of muscle.
- Ethanol 140 proof (Pharmco by Greenfield Global, catalog number: 111000140)
- Ethanol 200 proof (Pharmco by Greenfield Global, catalog number: 111000200 )
- Dulbecco’s Phosphate Buffered Saline (DPBS) (Corning, catalog number: 21-031-CV )
- Collagenase P (100 mg) (Roche, catalog number: 11 213 857 001)
- Dispase II (neutral protease, grade II) (Roche, catalog number: 04 942 078 001)
- CaCl2 (Sigma, catalog number: C1016-100G)
- NaCl (Fisher Scientific, catalog number: S271 )
- Dulbecco’s Modification Eagles Medium (DMEM) (Corning, catalog number: 15-013-CV )
- Dimethyl sulfoxide (DMSO) (Fisher BioReagent, catalog number BP213-100)
- Fetal Bovine Serum (FBS) (Gibco, catalog number: 10437-028 )
- Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco, catalog number: 15140-122 )
- GlutaMAX supplement (Thermo Fisher Scientific, Gibco, catalog number: 35050-061 )
- Stericup Quick release-GP sterile vacuum filtration system (Millipore, catalog number: S2GPU05RE)
- Triton X-100 solution (10%) (Sigma, catalog number: 93443 )
- TrypLE Express Enzyme (no phenol red) (Thermo Fisher Scientific, Gibco, catalog number: 12604-013 )
- BCA protein assay kit (Thermo Fisher Scientific, Pierce, catalog number: 23225 )
- Criterion TGX stain-free protein gel
10% 18-well, midi-gel (Bio-Rad, catalog number: 5678034)
12% 18-well, midi-gel (Bio-Rad, catalog number: 5678044)
10% 10-well, mini-gel (Bio-Rad, catalog number: 4568033)
12% 10-well, mini-gel (Bio-Rad, catalog number: 4568043) - Precision Plus Protein, all blue pre-stained protein standards (Bio-Rad, catalog number: 1610373 )
- Precision Plus Protein, Unstained protein standards (Bio-Rad, catalog number: 1610363 )
- Tris base (Bio-Rad, catalog number: 1610719 )
- Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144S-500 )
- Glycine (Bio-Rad, catalog number: 1610718 )
- Sodium dodecyl sulphate (SDS) (Bio-Rad, catalog number: 64086876)
- Laemmli sample buffer (4x) (Bio-Rad, catalog number: 1610747 )
- Dithiothreitol (DTT) (Sigma/Millipore, Roche, catalog number: 10708984001 )
- Methanol (Fisher Scientific, catalog number: A4121)
- Non-fat dry milk (Labscientific, catalog number: M0842)
- Bovine Serum Albumin (BSA) Fraction V (Roche, catalog number: 03116964001)
- Primary and secondary antibodies (Table 1)
Table 1. Primary and secondary antibodies
- Sucrose (Fisher Scientific, Fisher Chemical, catalog number: S5-500 )
- cOmplete protease inhibitor cocktail (Millipore/Sigma, Roche, catalog number: 11697498001 )
- 1.0 M Magnesium acetate solution (Mg(Ac)2) (Sigma, catalog number: 63052-100ml)
- Trichloroacetic Acid (TCA) (Sigma, catalog number: 91228-100G)
- Tween-20 (10%) (Bio-Rad, catalog number: 161-0781)
- SuperSignal west Femto (Thermo Fisher Scientific, catalog number: 34096 )
- Collagenase P stock solution (4x) (see Recipes)
- Dispase II stock solution (10x) (see Recipes)
- CaCl2 (1 M) (see Recipes)
- Digestion mixture (see Recipes)
- Complete medium (see Recipes)
- Freezing medium (see Recipes)
- Exosome free FBS (see Recipes)
- Complete exosome free medium (see Recipes)
- 0.1% Triton X-100 (see Recipes)
- BCA reagent working solution (see Recipes)
- 1.0 M DTT (see Recipes)
- 0.5 M Tris-HCl, pH 7.4 (see Recipes)
- Protease inhibitor stock solution (100x) (see Recipes)
- 10 mM Tris-HCl pH 7.4/1 mM Mg(Ac)2 (see Recipes)
- Sucrose gradient working solution (11 ml per sample) (see Recipes)
- 100% TCA (see Recipes)
- 80% ethanol (see Recipes)
- Western blot running buffer (10x) (see Recipes)
- Western blot running buffer (1x) (see Recipes)
- Transfer buffer (10x) (see Recipes)
- Transfer buffer (1x) (see Recipes)
- 20x Tris-buffered saline (TBS) (see Recipes)
- TBS-T(1 L) (see Recipes)
- Blocking buffer (see Recipes)
- 3% BSA-TBS-T (see Recipes)
- SuperSignal west Femto solution (per membrane) (see Recipes)
Equipment
- Aluminum dissection pan with vinyl dissecting pad (Carolina, catalog number: 629004)
- Dissection tools: sterilized scissors and forceps (Roboz, catalog numbers: RS-5983, RS-5877, RS-5358, RS-5135 )
- O2/CO2 incubator (Sanyo, catalog number: MC0-18M)
- Biosafety hood 1300 series A2 (Thermo Scientific, model: 1377)
- Water bath Isotemp 220 (Fisher Scientific, catalog number: FS220)
- Ice bucket (no particular brand is required)
- Centrifuge 5430R with rotor FA-35-6-30 (Eppendorf, catalog number: 022620659) and rotor 5430/R FA-45-48-11 (Eppendorf, catalog number: 5427754008)
- Countess automated cell counter (Thermo Fisher Scientific, Invitrogen, catalog number: C10227 )
- Ultra-centrifuge Optima XPM (Beckman Coulter, catalog number: A99842)
- Alphapette Pipettors
0.5-10 μl (Midwest Scientific, catalog number: ASALPHA A-10)
2-20 μl (Midwest Scientific, catalog number: ASALPHA A-20)
20-200 μl (Midwest Scientific, catalog number: ASALPHA A-200)
100-1,000 μl (Midwest Scientific, catalog number: A-1000 ) - Pipetboy acu 2 (Fisher Scientific, Integra Biosciences Corp, catalog number :155015)
- Criterion vertical electrophoresis cell (midi) (Bio-Rad, catalog number: NC0165100 or 1656001 )
- Mini-Protean tetra vertical electrophoresis cell (Bio-Rad, catalog number: 1658004 )
- PowerPac HC High-current power supply (Bio-Rad, catalog number: 1645052 )
- Chemidoc MP imaging system (Bio-Rad, catalog number: 12003154)
- Mini Trans-Blot electrophoretic transfer cell (Bio-Rad, catalog number: 1703930 )
- Criterion Blotter with plate electrodes (Bio-Rad, catalog number: 1704070 )
- Incubation containers for Western blots; the lids of pipet tip racks
- Tube rack for 50 ml tubes
- Fluostar Omega multi-mode microplate reader (BMG Labtech)
- Plexiglass (in house made)
- Belly dancer shaker (IBI scientific, catalog number: BDRAA115S)
- Stirrer (Fisher Scientific, Isotemp, catalog number: 11-100-49S )
- Stirring bar (Fisher Scientific, catalog number: 50-999-347)
Software
- Image Lab, Version 6.0.1 build 34 (2017) (Bio-Rad)
- Omega, Version 3.00R2 (BMG Labtech)
- Omega Analysis, Version 2.40, Build 13 (BMG Labtech)
Procedure
文章信息
版权信息
© 2020 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
van de Vlekkert, D., Qiu, X., Annunziata, I. and d'Azzo, A. (2020). Isolation, Purification and Characterization of Exosomes from Fibroblast Cultures of Skeletal Muscle. Bio-protocol 10(7): e3576. DOI: 10.21769/BioProtoc.3576.
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分类
细胞生物学 > 细胞器分离 > 外来体
细胞生物学 > 细胞分离和培养 > 单层培养
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