Tissue types | Collection and pre-processing | EV characterization | Method of analysis | Key findings | Citation | Author | 影響因子 | 備注 |
組織處理 | Pre-EV isolation | EV-isolation | Methods | Markers | PMID | Year |
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新鮮人/動(dòng)物組織 |
| 2500g, 30min; 10000g, 35min; | 0.2 μm過(guò)濾; 110000g, 2h; | NTA, WB, TEM, IEM, fluorescence staining, FCM | CD9, CD63, CD8, ESCRT proteins (Alix, TSG101) | / | EVs分離鑒定方法; | 27801511 | 2016 |
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人轉(zhuǎn)移性黑色素瘤組織 | 新鮮組織切碎到1-2mm; 在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培養(yǎng)基中37°C孵育30min; | 70 μm過(guò)濾; 300g, 10min; 2000g, 20min; 16500g, 20min; | 118000g, 2.5h; | TEM, NTA, WB | CD9, CD63, CD81, calnexin, flotillin-1 | NanoLC-MS/MS analysis | 分離EVs亞型方法; ADAM10富集于LD EVs; mitofilin富集于大EVs; | 32128073 | 2020 | Crescitelli et al. |
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人黑色素瘤組織 | 新鮮組織切碎到2mm; 在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培養(yǎng)基中37°C孵育30min,期間24rpm緩慢振蕩; | 300g, 10min; 2000g, 20min; 16500g, 20min; | 118000g, 2.5h; 碘克沙醇密度梯度離心, 186000g, 16h; | TEM, WB, ExoView analysis | CD63, CD81, CD9, flotillin-1, Calnexin | RNA profile analysis, ExoView, proteomic analysis | 人黑色素瘤組織外泌體分離鑒定方案; | 33495626 | 2021 | Crescitelli et al. |
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新鮮人腸組織 | 組織切碎到<0.5cm; 含collagenase I(300U/ml)的HBSS中孵育30min,期間緩慢振蕩; 用含有蛋白酶抑制劑的PBS終止消化; | 70 μm過(guò)濾; 1000g, 10min; 2000g, 20min; 5000g, 30min; 15000g, 1h; 0.2 μm PVDF過(guò)濾; | 120000g, 130min; 碘克沙醇密度梯度離心, 288000g, 5h; 100000g, 70min; | NTA, TEM, WB | CD9, CD63, CD81, Alix, Tsg101 | RT-qPCR | 分離腸組織外泌體并應(yīng)用于腸缺血-再灌注(I/R)損傷研究; | 32090587 | 2020 |
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人腎癌及癌旁組織 | 組織切碎; 加入4ml DMEM,4°C孵育1h; | 2000g, 20min; 16000g, 20min; | 100000g, 90min; 100000g, 90min; | WB, TEM, NTA | CD9, CD63, CD81 | Quantitative LC/MS analysis | Te‐EVs; 蛋白組分析; AZU1 | 28975613 | 2018 |
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人轉(zhuǎn)移性黑色素瘤組織 | 組織切碎到1-2mm; 在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培養(yǎng)基中37°C孵育30min; | 70 μm過(guò)濾; 300g, 10min; 2000g, 20min; 16500g, 20min; | 110000g, 2.5h; | TEM, WB, ELISA, particle measurement | CD9, CD81 | MS analysis, RNA detection | 黑色素瘤組織外泌體富含線(xiàn)粒體膜蛋白; | 31497264 | 2019 |
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尸檢腦組織 | 組織冰上切碎,冰上解凍; 在含有collagenase III (75 U/mL)的Hibernate-E培養(yǎng)基中37°C孵育20min,期間緩慢振蕩; 加入蛋白酶抑制劑(PI/PS)終止消化; | 37°C水浴振蕩20min; 300g, 5min; 2000g, 10min; 10000g, 5min; | Triple sucrose cushion; 180000g, 3h; | TEM, WB, IB | Calnexin | Small RNA sequencing | AD腦組織EVs miRNA分析; | 32922692 | 2020 |
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毫米大小癌及癌旁組織 | 組織切碎; 在含有雙抗的RPMI1640培養(yǎng)基中37°C孵育24h; | 500g, 10min; 3000g, 20min; 12000g, 20min; | 100000g, 70min; 1 ml sucrose density cushion, 100000g, 70min; | WB, TEM, NTA | CD9, CD81, TSG101 | MS蛋白組 | pan-EVP markers (ACTB, MSN, RAP1B); 腫瘤特異性EVP蛋白; | 32795414 | 2020 |
| 66 |
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小鼠黑色素瘤組織 | 組織切碎到<3mm, PBS洗一遍; 500g, 4min; 組織解離為單細(xì)胞懸液(含有25 μg/ml DNase I, 5 Wünsch units of Liberase的PBS,37°C,20min,緩慢振蕩); 70μm過(guò)濾; PBS(含5 mM EDTA,25 μg/ml DNase I)洗一遍; | 500g, 10min; 500g, 10min; 2000g, 15min; 2000g, 15min; | 16.5K EVs: 16500g, 24min (×2); 100K EVs: 100000g,70min (×2); | TEM, WB | Calnexin, cytochrome-C, GM130 | LC-MS/MS, transcriptomics analysis | Cells from two types of melanoma phenocopy migratory behaviour through EV exchange. | 29907695 | 2018 |
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結(jié)直腸癌及癌旁組織 | 新鮮組織冰上切碎; 在含有collagenase I (250 units/ml) RPMI1640 培養(yǎng)基中37°C孵育30min; 置于冰上,加入含多種蛋白酶抑制劑的PBS; 吹散細(xì)胞; | 60mm篩網(wǎng); 40mm篩網(wǎng); 400g, 10min; 2000g, 20min; 15000g, 40min; 0.22 μm PES過(guò)濾; | 120000g, 4h (×2); 碘克沙醇密度梯度離心; | IB | CD63, CD81, Syntenin-1, Calreticulin | MS, RNA sequencing, DNA analysis, direct immunoaffinity Capture | CD9, CD63, CD81, Annexin V在外泌體中缺失; Annexin A1和A2為非外泌體EVs新markers; | 30951670 | 2019 |
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小鼠結(jié)直腸癌組織 | PBS洗一遍; 組織在含2 mM EDTA的PBS中切碎; | 70μm細(xì)胞篩網(wǎng); 500g, 5min; 3000g, 10min; 10000g, 30min; | 110000g, 70min; 134000g, 70min; | TEM, NTA, WB, Flow cytometry | CD9, CD81, ALIX | LC-MS/MS, miRNA profiling, Lipidome analysis | TAM-EVs誘導(dǎo)炎癥及抗腫瘤反應(yīng); | 31167148 | 2019 |
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AD小鼠腦組織 | 在Hibernate-E培養(yǎng)基中37°C孵育20min; 研磨(loose-fit Dounce homogenizer); | 500g, 5min; 2000g, 10min; 10000g, 30min; 0.45 μm 過(guò)濾; | 100000g, 2h; 1.5 ml of 0.25 M sucrose buffer for gradient purification; floatation density gradient. | WB | CD63, CD81, Alix, TSG101, HSC70, Rab8a | MS, enrichment analysis | Ti-EVs分離方法; | 29894726 | 2018 |
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人腦組織 | 在Hibernate-E培養(yǎng)基(木瓜蛋白酶,20 units/ml)中37°C孵育15min; | 40 μm 篩網(wǎng); 300g, 10min; 2000g, 10min; 10000g, 10min; 0.22 μm 過(guò)濾; | 100000g, 70min; 0.475 M of sucrose solution | NTA, TEM | / | Label-free Nano-LC-MS/MS analysis | Tau、Aβ1-42上調(diào); | 32301581 | 2020 |
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人腦組織 | PBS中切碎,渦旋; | 300g, 10min; 1200g, 10min(×2); 0.22 μm 過(guò)濾; 10000g, 30min(×2); | 22000g, 22h; | WB, TEM, Immunogold Labeling, | CD63, GAPDH, flotillin-2 | MiRNA expression analysis, qPCR analysis | SZ樣品miR-497上調(diào); BD樣品miR-29c上調(diào); | 23382797 | 2013 |
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恒河猴腦組織 | 在Hibernate-A培養(yǎng)基(木瓜蛋白酶,20 units/ml)中37°C振蕩孵育15min; | 40 μm 篩網(wǎng); 5 μm 篩網(wǎng); 0.22 μm 篩網(wǎng); 300g, 10min; 2000g, 10min; 10000g, 30min; | 100000g,60min (×2); 2 ml of 0.95 M sucrose solution and inserted inside a sucrose step gradient column; 200000g,16h; | TEM, WB | CD9, CD63, CD81, HSP70, flotillin, TSG101 | Small RNA sequencing, qRT-PCR | Neurotoxicity triggered by EV-miR-21 was not influenced by apoptosis inhibition but restricted by necrostatin-1. | 26154133 | 2015 |
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小鼠腦組織 |
| 40 μm 篩網(wǎng); 0.2 μm 篩網(wǎng); 300g, 10min; 2000g, 10min; 10000g, 30min; | 100000g,70min (×2); 2 ml of 0.95 M sucrose solution centrifuged at 200000g for 16 h. | Immunoelectron microscopy | TSG101 | Gene expression analysis, image analysis | 小神經(jīng)膠質(zhì)細(xì)胞通過(guò)外泌體擴(kuò)散tau; | 26436904 | 2015 |
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人腦組織 | 冰凍組織冰上切碎; 在Hibernate-E培養(yǎng)基(collagenase III ,75 U/ml )中37°C振蕩孵育20min; 蛋白酶抑制劑終止消化(PhosSTOP和Complete Protease Inhibitor); | 300g, 15min; 2000g, 15min; 0.2 μm 篩網(wǎng); 10000g, 30min; | Sucrose density gradient ultracentrifugation (SDGU); 110000g,70min; ultrafiltration through a 10 kDa MWCO protein concentrator. | TEM, NTA, WB, NanoFCM flow analysis | CD9, CD63, CD81, Rab27, TSG101, Syntenin, Calnexin, GM130 | Small RNA sequencing, MS | 3個(gè)物種腦組織Ti-EVs分離比較,強(qiáng)調(diào)了不同物種的分離參數(shù); | 32944174 | 2020 |
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人脂肪組織 |
| 800g, 10min; 2000g, 10min; 12000g, 30min; 0.2 μm 篩網(wǎng); | 100000g, 2h; | Fluorescence NTA, WB, EM | CD9, CD63, TSG101, Grp94 (a negative control) | MS analysis, Ingenuity pathway analysis | Adipose Ti-EVs mediate changes in placental functions in GDM and are involved in some pregnancy complications. | 30517676 | 2019 |
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脂肪組織 | 切碎至1-2mm; 加入到含雙抗的α-MEM培養(yǎng)基中,37°C 100rpm/min振蕩孵育2天; | 2000g, 10min; 5000g, 30min; | 5000g, 30min; Exosome Isolation TM reagent 4°C孵育過(guò)夜; 10000g, 1h; | WB, TEM, Zetasizer Nano ZS | CD9, CD63, ALIX, TSG101 | MS analysis, Bioinformatic analysis, Real-time PCR | NPM3, STEAP3, DAD1與脂肪形成 | 32597661 | 2020 |
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Human obese white adipose tissue explant |
| 1800g, 5min; 0.22 μm 篩網(wǎng); 10000g, 20min; | 100000g,90min (×2); | TEM, NTA, IB | CD9, CD63, CD81, negative control GRP94 | MS DDA qualitative analysis | Obese AT release functional EVs carrying AT and obesity-specific biomarkers depending on the original AT. | 33465489 | 2022 |
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小鼠脂肪組織 | 切碎至>4mm; 加入到含抗生素和10%FBS(已去除外泌體)的DMEM培養(yǎng)基中,37°C 培養(yǎng); | 200g, 10min; 500g, 10min(×2); 2000g, 15min; 10000g, 30min; | 70000g, 60min; 5 ml of 2.6 M sucrose at 270000g 16 h; Gradient fractions were centrifugated at 70000g 1h. | / | / | Fluorescence activated cell sorter (FACS) analysis | Adipose Ti-ELVs mediate crosstalk between AT and macrophages; ObELV induced TNF-α and IL-6 activation and insulin resistance require the TLR4/TRIF pathway. | 19675137 | 2009 |
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小鼠脂肪組織 | 灌洗以去除組織中血液; 切碎; 37°C解離組織1h(100 mM HEPES, 1.5% BSA, 5 mM glucose, 1 mM calcium and 1mg/ml collagenase D, 2.4U/ml dispase II); 2 mM EGTA中止消化; | 100 μm 篩網(wǎng); 600g, 5min; 1200g, 15min; 10000g, 15min; 0.22 μm 篩網(wǎng); | 100000g, 90min (×2); | NTA, TEM, WB | CD63, Alix, TSG101 | Proteomics analysis, LC‐MS lipidomics analysis. | Adipose Ti‐EVs are involved in the response to changes in systemic nutrient conditions. | 30293865 | 2018 | 66 |
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小鼠脂肪組織 | 剪碎; 1000g,5min,清洗組織; 培養(yǎng)基中37?°C孵育30min; 換液,培養(yǎng)基中37?°C孵育2h以釋放外泌體; | 1000g for 5 min at room temperature and re‐dissolved in medium; incubated for 30 min at 37°C, 5% CO2. | Medium exchange, tissues were incubated for 2 h at 37°C, 5% CO2. The supernatant was used for EVs isolation according to the manufacturer's instruction. | WB, TEM | CD63, Hsp70, CytoC, α‐Tubulin | MiRNA profiling | 外泌體miR‐92a與人BAT活性負(fù)相關(guān); | 27117818 | 2016 |
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小鼠肝組織 | 通過(guò)灌注膠原酶來(lái)解離肝臟組織; | 肝灌注; 70 μm 篩網(wǎng); 50g, 10min; 300g, 10min; 2000g, 20min; 10000g, 70min; | 100000g, 70min (×2); | NTA, tunable resistive pulse sensing | / | / | An optimum and replicable procedure for the isolation of hepatic Ti‐EVs. | 31498323 | 2019 |
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| JOVE |