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上海研匠生物科技有限公司
组织外泌体提取
每样本
Tissue types | Collection and pre-processing | EV characterization | Method of analysis | Key findings | Citation | Author | 影响因子 | 备注 | ||||
组织处理 | Pre-EV isolation | EV-isolation | Methods | Markers | PMID | Year | ||||||
新鲜人/动物组织 | 2500g, 30min; 10000g, 35min; |
0.2 μm过滤; 110000g, 2h; |
NTA, WB, TEM, IEM, fluorescence staining, FCM | CD9, CD63, CD8, ESCRT proteins (Alix, TSG101) |
/ | EVs分离鉴定方法; | 27801511 | 2016 | ||||
人转移性黑色素瘤组织 | 新鲜组织切碎到1-2mm; 在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min; |
70 μm过滤; 300g, 10min; 2000g, 20min; 16500g, 20min; |
118000g, 2.5h; | TEM, NTA, WB | CD9, CD63, CD81, calnexin, flotillin-1 | NanoLC-MS/MS analysis | 分离EVs亚型方法; ADAM10富集于LD EVs; mitofilin富集于大EVs; |
32128073 | 2020 | Crescitelli et al. | ||
人黑色素瘤组织 | 新鲜组织切碎到2mm; 在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min,期间24rpm缓慢振荡; |
300g, 10min; 2000g, 20min; 16500g, 20min; |
118000g, 2.5h; 碘克沙醇密度梯度离心, 186000g, 16h; |
TEM, WB, ExoView analysis | CD63, CD81, CD9, flotillin-1, Calnexin | RNA profile analysis, ExoView, proteomic analysis | 人黑色素瘤组织外泌体分离鉴定方案; | 33495626 | 2021 | Crescitelli et al. | ||
新鲜人肠组织 | 组织切碎到<0.5cm; 含collagenase I(300U/ml)的HBSS中孵育30min,期间缓慢振荡; 用含有蛋白酶抑制剂的PBS终止消化; |
70 μm过滤; 1000g, 10min; 2000g, 20min; 5000g, 30min; 15000g, 1h; 0.2 μm PVDF过滤; |
120000g, 130min; 碘克沙醇密度梯度离心, 288000g, 5h; 100000g, 70min; |
NTA, TEM, WB | CD9, CD63, CD81, Alix, Tsg101 | RT-qPCR | 分离肠组织外泌体并应用于肠缺血-再灌注(I/R)损伤研究; | 32090587 | 2020 | |||
人肾癌及癌旁组织 | 组织切碎; 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; 蛋白组分析; AZU1 |
28975613 | 2018 | |||
人转移性黑色素瘤组织 | 组织切碎到1-2mm; 在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min; |
70 μm过滤; 300g, 10min; 2000g, 20min; 16500g, 20min; |
110000g, 2.5h; | TEM, WB, ELISA, particle measurement | CD9, CD81 | MS analysis, RNA detection | 黑色素瘤组织外泌体富含线粒体膜蛋白; | 31497264 | 2019 | |||
尸检脑组织 | 组织冰上切碎,冰上解冻; 在含有collagenase III (75 U/mL)的Hibernate-E培养基中37°C孵育20min,期间缓慢振荡; 加入蛋白酶抑制剂(PI/PS)终止消化; |
37°C水浴振荡20min; 300g, 5min; 2000g, 10min; 10000g, 5min; |
Triple sucrose cushion; 180000g, 3h; |
TEM, WB, IB | Calnexin | Small RNA sequencing | AD脑组织EVs miRNA分析; | 32922692 | 2020 | |||
毫米大小癌及癌旁组织 | 组织切碎; 在含有双抗的RPMI1640培养基中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蛋白组 | pan-EVP markers (ACTB, MSN, RAP1B); 肿瘤特异性EVP蛋白; |
32795414 | 2020 | 66 | ||
小鼠黑色素瘤组织 | 组织切碎到<3mm, PBS洗一遍; 500g, 4min; 组织解离为单细胞悬液(含有25 μg/ml DNase I, 5 Wünsch units of Liberase的PBS,37°C,20min,缓慢振荡); 70μm过滤; 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 | |||
结直肠癌及癌旁组织 | 新鲜组织冰上切碎; 在含有collagenase I (250 units/ml) RPMI1640 培养基中37°C孵育30min; 置于冰上,加入含多种蛋白酶抑制剂的PBS; 吹散细胞; |
60mm筛网; 40mm筛网; 400g, 10min; 2000g, 20min; 15000g, 40min; 0.22 μm PES过滤; |
120000g, 4h (×2); 碘克沙醇密度梯度离心; |
IB | CD63, CD81, Syntenin-1, Calreticulin | MS, RNA sequencing, DNA analysis, direct immunoaffinity Capture | CD9, CD63, CD81, Annexin V在外泌体中缺失; Annexin A1和A2为非外泌体EVs新markers; |
30951670 | 2019 | 66 | ||
小鼠结直肠癌组织 | PBS洗一遍; 组织在含2 mM EDTA的PBS中切碎; |
70μm细胞筛网; 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诱导炎症及抗肿瘤反应; | 31167148 | 2019 | |||
AD小鼠脑组织 | 在Hibernate-E培养基中37°C孵育20min; 研磨(loose-fit Dounce homogenizer); |
500g, 5min; 2000g, 10min; 10000g, 30min; 0.45 μm 过滤; |
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分离方法; | 29894726 | 2018 | |||
人脑组织 | 在Hibernate-E培养基(木瓜蛋白酶,20 units/ml)中37°C孵育15min; | 40 μm 筛网; 300g, 10min; 2000g, 10min; 10000g, 10min; 0.22 μm 过滤; |
100000g, 70min; 0.475 M of sucrose solution |
NTA, TEM | / | Label-free Nano-LC-MS/MS analysis | Tau、Aβ1-42上调; | 32301581 | 2020 | |||
人脑组织 | PBS中切碎,涡旋; | 300g, 10min; 1200g, 10min(×2); 0.22 μm 过滤; 10000g, 30min(×2); |
22000g, 22h; | WB, TEM, Immunogold Labeling, | CD63, GAPDH, flotillin-2 | MiRNA expression analysis, qPCR analysis | SZ样品miR-497上调; BD样品miR-29c上调; |
23382797 | 2013 | |||
恒河猴脑组织 | 在Hibernate-A培养基(木瓜蛋白酶,20 units/ml)中37°C振荡孵育15min; | 40 μm 筛网; 5 μm 筛网; 0.22 μm 筛网; 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 | |||
小鼠脑组织 | 40 μm 筛网; 0.2 μm 筛网; 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 | 小神经胶质细胞通过外泌体扩散tau; | 26436904 | 2015 | 28 | |||
人脑组织 | 冰冻组织冰上切碎; 在Hibernate-E培养基(collagenase III ,75 U/ml )中37°C振荡孵育20min; 蛋白酶抑制剂终止消化(PhosSTOP和Complete Protease Inhibitor); |
300g, 15min; 2000g, 15min; 0.2 μm 筛网; 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个物种脑组织Ti-EVs分离比较,强调了不同物种的分离参数; | 32944174 | 2020 | |||
人脂肪组织 | 800g, 10min; 2000g, 10min; 12000g, 30min; 0.2 μm 筛网; |
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 | ||||
脂肪组织 | 切碎至1-2mm; 加入到含双抗的α-MEM培养基中,37°C 100rpm/min振荡孵育2天; |
2000g, 10min; 5000g, 30min; |
5000g, 30min; Exosome Isolation TM reagent 4°C孵育过夜; 10000g, 1h; |
WB, TEM, Zetasizer Nano ZS | CD9, CD63, ALIX, TSG101 | MS analysis, Bioinformatic analysis, Real-time PCR | NPM3, STEAP3, DAD1与脂肪形成 | 32597661 | 2020 | |||
Human obese white adipose tissue explant | 1800g, 5min; 0.22 μm 筛网; 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 | ||||
小鼠脂肪组织 | 切碎至>4mm; 加入到含抗生素和10%FBS(已去除外泌体)的DMEM培养基中,37°C 培养; |
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 | |||
小鼠脂肪组织 | 灌洗以去除组织中血液; 切碎; 37°C解离组织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 筛网; 600g, 5min; 1200g, 15min; 10000g, 15min; 0.22 μm 筛网; |
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 | ||
小鼠脂肪组织 | 剪碎; 1000g,5min,清洗组织; 培养基中37 °C孵育30min; 换液,培养基中37 °C孵育2h以释放外泌体; |
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 | 外泌体miR‐92a与人BAT活性负相关; | 27117818 | 2016 | |||
小鼠肝组织 | 通过灌注胶原酶来解离肝脏组织; | 肝灌注; 70 μm 筛网; 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 | JOVE |
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