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組織外泌體提取

2023-04-12 14:20:29 279

一般,組織外泌體提取的方法包括三個(gè)過(guò)程。首先,組織切成小塊,使組織具有更大的表面積。其次,在細胞培養基中利用組織解離酶與組織切片孵育,目的是使組織外泌體擴散到培養基中。最后,外泌體通過(guò)密度梯度離心、分子尺寸排阻等方法從培養基中分離出來(lái)。需要注意的是組織切片和組織消化過(guò)程中避免細胞的破碎,因為細胞破碎后會(huì )有大量的囊泡產(chǎn)生。


外泌體膜蛋白組質(zhì)譜鑒定與分析

對EVs表面的膜蛋白進(jìn)行生物素標記,然后利用鏈霉親和素磁珠進(jìn)行EVs膜蛋白的富集,再進(jìn)行EVs膜蛋白的質(zhì)譜鑒定分析,從而實(shí)現低豐度EVs膜蛋白的篩選。針對篩選的EVs膜蛋白,可以利用免疫膠體金電鏡進(jìn)一步驗證


外泌體膜蛋白芯片分析

利用定制化的膜蛋白抗體芯片直接對樣本中的外泌體進(jìn)行捕獲和檢測,無(wú)需進(jìn)行外泌體的提取。


外泌體蛋白質(zhì)組學(xué)分析(Label-Free)


蛋白質(zhì)組學(xué)(Proteomics)是指利用高分辨的蛋白質(zhì)分離技術(shù)和高效的蛋白質(zhì)鑒定技術(shù)在蛋白質(zhì)水平上整體性、動(dòng)態(tài)和定量地研究生命現象及規律的科學(xué)。外泌體蛋白組學(xué)即分析外泌體中所有蛋白質(zhì)的集合。

質(zhì)譜分析技術(shù)有著(zhù)高靈敏度,高精準度等特點(diǎn),能夠快速準確地分析外泌體蛋白,適用于外泌體蛋白組學(xué)的研究。非標定量法(Label-Free)是通過(guò)比較質(zhì)譜分析次數或質(zhì)譜峰強度,分析不同來(lái)源樣品蛋白的數量變化,認為肽段在質(zhì)譜中被捕獲檢測的頻率與其在混合物中的豐度正相關(guān),因此蛋白質(zhì)被質(zhì)譜檢測的計數反映了蛋白質(zhì)的豐度,通過(guò)適當的數學(xué)公式可以將質(zhì)譜檢測計數與蛋白質(zhì)的量聯(lián)系起來(lái),從而對蛋白質(zhì)進(jìn)行定量。


外泌體非編碼RNA組學(xué)

非編碼RNA(Non-coding RNA)是指不編碼蛋白質(zhì)的RNA,包括miRNA、lncRNA、circRNA、piRNA等。非編碼RNA發(fā)揮功能的方式很多,可以與蛋白、DNA和RNA相互作用,參與多種細胞活動(dòng),主要包括基因的激活和沉默,RNA的剪接、修飾和編輯,蛋白質(zhì)的翻譯等。

Tissue typesCollection and pre-processingEV characterizationMethod of analysisKey findingsCitationAuthor影響因子備注
組織處理Pre-EV isolationEV-isolationMethodsMarkersPMIDYear


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, FCMCD9, CD63, CD8,
ESCRT proteins (Alix, TSG101)
/EVs分離鑒定方法;278015112016


人轉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-1NanoLC-MS/MS analysis分離EVs亞型方法;
ADAM10富集于LD EVs;
mitofilin富集于大EVs;
321280732020Crescitelli et al.

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 analysisCD63, CD81, CD9, flotillin-1, CalnexinRNA profile analysis, ExoView, proteomic analysis人黑色素瘤組織外泌體分離鑒定方案;334956262021Crescitelli et al.

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, WBCD9, CD63, CD81, Alix, Tsg101RT-qPCR分離腸組織外泌體并應用于腸缺血-再灌注(I/R)損傷研究;320905872020


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, NTACD9, CD63, CD81Quantitative LC/MS analysisTe‐EVs;
蛋白組分析;
AZU1
289756132018


人轉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 measurementCD9, CD81MS analysis, RNA detection黑色素瘤組織外泌體富含線(xiàn)粒體膜蛋白;314972642019


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, IBCalnexinSmall RNA sequencingAD腦組織EVs miRNA分析;329226922020


毫米大小癌及癌旁組織組織切碎;
在含有雙抗的RPMI1640培養基中37°C孵育24h;
500g, 10min;
3000g, 20min;
12000g, 20min;
100000g, 70min;
1 ml sucrose density cushion,
100000g, 70min;
WB, TEM, NTACD9, CD81, TSG101MS蛋白組pan-EVP markers (ACTB, MSN, RAP1B);
腫瘤特異性EVP蛋白;
327954142020
66
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, WBCalnexin, cytochrome-C, GM130LC-MS/MS, transcriptomics analysisCells from two types of melanoma phenocopy migratory behaviour through EV exchange.299076952018


結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;
IBCD63, CD81, Syntenin-1, CalreticulinMS, RNA sequencing, DNA analysis, direct immunoaffinity CaptureCD9, CD63, CD81, Annexin V在外泌體中缺失;
Annexin A1和A2為非外泌體EVs新markers;
309516702019
66
小鼠結直腸癌組織PBS洗一遍;
組織在含2 mM EDTA的PBS中切碎;
70μm細胞篩網(wǎng);
500g, 5min;
3000g, 10min;
10000g, 30min;
110000g, 70min;
134000g, 70min;
TEM, NTA, WB, Flow cytometryCD9, CD81, ALIXLC-MS/MS, miRNA profiling, Lipidome analysisTAM-EVs誘導炎癥及抗腫瘤反應;311671482019


AD小鼠腦組織在Hibernate-E培養基中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.
WBCD63, CD81, Alix, TSG101, HSC70, Rab8aMS, enrichment analysisTi-EVs分離方法;298947262018


人腦組織在Hibernate-E培養基(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 analysisTau、Aβ1-42上調;323015812020


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-2MiRNA expression analysis, qPCR analysisSZ樣品miR-497上調;
BD樣品miR-29c上調;
233827972013


恒河猴腦組織在Hibernate-A培養基(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, WBCD9, CD63, CD81, HSP70, flotillin, TSG101Small RNA sequencing, qRT-PCRNeurotoxicity triggered by EV-miR-21 was not influenced by apoptosis inhibition but restricted by necrostatin-1.261541332015


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 microscopyTSG101Gene expression analysis, image analysis小神經(jīng)膠質(zhì)細胞通過(guò)外泌體擴散tau;264369042015
28
人腦組織冰凍組織冰上切碎;
在Hibernate-E培養基(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 analysisCD9, CD63, CD81, Rab27, TSG101, Syntenin, Calnexin, GM130Small RNA sequencing, MS3個(gè)物種腦組織Ti-EVs分離比較,強調了不同物種的分離參數;329441742020


人脂肪組織
800g, 10min;
2000g, 10min;
12000g, 30min;
0.2 μm 篩網(wǎng);
100000g, 2h;Fluorescence NTA, WB, EMCD9, CD63, TSG101, Grp94 (a negative control)MS analysis, Ingenuity pathway analysisAdipose Ti-EVs mediate changes in placental functions in GDM and are involved in some pregnancy complications.305176762019


脂肪組織切碎至1-2mm;
加入到含雙抗的α-MEM培養基中,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 ZSCD9, CD63, ALIX, TSG101MS analysis, Bioinformatic analysis, Real-time PCRNPM3, STEAP3, DAD1與脂肪形成325976612020


Human obese white adipose tissue explant
1800g, 5min;
0.22 μm 篩網(wǎng);
10000g, 20min;
100000g,90min (×2);TEM, NTA, IBCD9, CD63, CD81, negative control GRP94MS DDA qualitative analysisObese AT release functional EVs carrying AT and obesity-specific biomarkers depending on the original AT.334654892022


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培養基中,37°C 培養;
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) analysisAdipose Ti-ELVs mediate crosstalk between AT and macrophages; ObELV induced TNF-α and IL-6 activation and insulin resistance require the TLR4/TRIF pathway.196751372009


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, WBCD63, Alix, TSG101Proteomics analysis, LC‐MS lipidomics analysis.Adipose Ti‐EVs are involved in the response to changes in systemic nutrient conditions.30293865201866

小鼠脂肪組織剪碎;
1000g,5min,清洗組織;
培養基中37?°C孵育30min;
換液,培養基中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, TEMCD63, Hsp70, CytoC, α‐TubulinMiRNA profiling外泌體miR‐92a與人BAT活性負相關(guān);271178182016


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.314983232019

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