Name: sample preparation and imaging of exosomes by transmission electron microscopy
Uploaded: 2018-01-04T13:00:00.000+00:00
Duration: 11 min 15 s
Description: Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

이 연구는 생물에 의해 지원 되었다 &#38; 의료 기술 개발 프로그램 국가 연구 재단 (NRF)의 &#38;는 정부에 의해 투자 (MSIP &#38; 보건복지부) (No. 2016M3A9B6904244). 우리는 또한 exosome의 정화의 약용 Bioconvergence 연구 센터의 회원을 감사합니다.

1. 블록 준비, 단면, 얼룩이 지 고는 Exosome의 이미징
<ol>
	<li>1.5 h23에 대 한 100000 x g에서 원심 분리에 의해 HCT116 세포의 문화 상쾌한에서 exosomes을 펠 렛. 표면에 뜨는 문화를 제거 하 고 신중 하 게 순화 exosome 펠 릿 1 mL의 0.1 M 나트륨 cacodylate 솔루션 (pH 7.0) 4 ° c.에 1 시간에에서 2.5%도 해결 0.1 M 나트륨 cacodylate, 160 ml 증류수 (DW) 4.28 g cacodylic 산 분해. 0.1 M HCl 최대 200 mL 증류수와 함께 게와 7.4에 pH를 조정 합니다.</li>
	<li>정착 액을 제거 하 고 실 온에서 0.1 M 나트륨 cacodylate 버퍼의 1 mL와 함께 알 약을 씻어. 각 변경 10 분 지속으로 세 번을 반복 합니다.</li>
	<li>후 4 ° c.에 2% 오스뮴 tetroxide 1 h의 1 mL와 함께 샘플을 수정</li>
	<li>정착 액을 제거 하 고 세 번 0.1 M 나트륨 cacodylate와 린스 버퍼 마다 10 분.</li>
	<li>등급된 아세톤 시리즈와 10 분 동안 품 어 (50%, 60%, 70%, 80%, 90%, 95%, 100%, 각각) 뿌리에.</li>
	<li>아세톤을 제거 하 고 30 분에 대 한 3:1 아세톤: 낮은 점도 포함 혼합물의 솔루션을 품 어. Exosome 펠 렛 튜브입니다.</li>
	<li>매체를 제거 하 고 1:1 아세톤: 낮은 점도 포함 혼합 매체, 추가 다음 30 분 동안 품 어.</li>
	<li>매체를 제거 하 고 1:3 아세톤: 낮은 점도 포함 혼합 매체, 추가 다음 30 분 동안 품 어.</li>
	<li>매체를 제거 하 고 추가 100% 낮은 점도 혼합물을 포함 하 고 하룻밤 실 온에서 품 어.</li>
	<li>65 ° c.에 24 h에 대 한 포함 형과 빵을 사용 하 여 혼합물을 포함 하는 순수한 낮은 점도 샘플 포함</li>
	<li>60 nm 두께 울트라-톰을 통해 섹션을 준비 합니다.</li>
	<li>더블-20 분 동안 2 %uranyl 아세테이트로 얼룩 그리고 10 분 시트르산. 레이놀즈 리드 솔루션에 대 한 총 50 mL 증 류 물 납 질 산 및 나트륨 시트르산의 1.76 g 1.33 g를 추가 합니다.</li>
	<li>전송 전자 현미경 검사 법 80에서 아래의 표에서 관찰 kV.</li>
	<li>"취득"을 클릭 하 고 클릭 "파일" 및 "이름으로 저장" CCD 카메라 시스템 80에서 전자 현미경에 kV. 노출 시간에 대 한 자동 설정을 따릅니다.</li>
</ol>
2. 면역-얼룩 섹션 및 이미징 (그림 1)의
<ol>
	<li>매우 톰을 사용 하 여 60 nm ultrathin 섹션을 잘라 고 니켈 격자에 수집 합니다.</li>
	<li>10 분 무료 알데하이드 그룹을 끄다 0.02 M 글리신의 50 µ L에서에서 격자를 품 어.</li>
	<li>린스에 DW의 100 μ 3 번 각각 1 %BSA 포함 하는 PBS에서 1 시간에 대 한 실 온에서 10 분 품.</li>
	<li>1 시간 (해당 되는 경우 필요한,이 단계 실행 되어야 한다 4 ° C에서 하룻밤.)에 대 한 격자에서 50-100 µ L의 KRS 항 체24 (1: 100 0.1 %BSA 포함 하는 PBS에) 안티를 품 어</li>
	<li>10 분 동안 0.1 %BSA 포함 하는 PBS의 5 별도 방울 (50 µ L)으로 격자를 씻어.</li>
	<li>1 h (안티-토끼 IgG 0.1 %BSA 포함 하는 PBS에 10 nm 금 입자 (1: 100)를 활용)에 대 한 2차 항 체의 방울을 격자를 전송 합니다.</li>
	<li>5 세척 격자 각 10 분 0.1 %BSA 포함 하는 PBS의 방울 (50 µ L)을 구분 합니다.</li>
	<li>더블-얼룩 어두운 조건 하에서 20 분 동안 2 %uranyl 아세테이트와 Reynold의 리드 시트르산 10 분, 각각.</li>
	<li>"취득"을 클릭 하 고 클릭 "파일" 및 "저장" CCD 카메라 시스템 80에서 가장 아래에 kV. 노출 시간에 따라 자동 설정.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56482/56482fig1.jpg"> 
그림 1: 샘플 준비 과정 immunostaining. (A) 이중 고정된 exosome 탈수 이며 저 점도 수 지 혼합물 포함으로 침투. (B) 포함 수 지. (C) 다이아몬드 칼으로 초박형 섹션. 면역-골드 라벨에 대 한 (D) 설정. Ultrathin 섹션 격자는 젖은 종이 타월로 배치 parafilm에 액체 방울에 배치 됩니다. 각 섹션은 50-100 µ L 항 체, 방울과 알을 품을 다음 버퍼로 세척. (E) uranyl 아세테이트와 얼룩 더블 고 시트르산. 뚜껑은 알루미늄 호 일 중 금속 얼룩으로 덮여 있다. (F) 솔루션 얼룩 필터 종이 의해 제거 됩니다. <a href="//ecsource.jove.com/files/ftp_upload/56482/56482fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
3. 부정적인 얼룩이 지기
<ol>
	<li>글로우 방전 발광 방 전자에 의해 1 분 동안 얇은 formvar/탄소 코팅 필름 200 메쉬 구리 EM 격자.</li>
	<li>2%의 1 mL와 함께 순화 exosomes 수정 Paraformaldehyde (PFA) 5 분. 주의: Paraformaldehyde 가스는 유독 합니다. 모든 작업은 통풍이 증기 두건에서 행해져야 한다.</li>
	<li>5-7 µ L exosome 정지 솔루션에 로드 하 고 1 분 동안 품 어. Exosome의 농도가 너무 높은 경우, 희석 농도를 1/2-1/5.</li>
	<li>주사기로 EM 격자의 표면에 필터링 된 1 %uranyl 아세테이트 (UA) 솔루션 ~ 20 방울과 함께 즉시 얼룩.</li>
	<li>필터 종이 그리드 가장자리에 연락 하 여 격자에 과잉 UA 솔루션을 제거 합니다.</li>
	<li>신속 하 게 물 한 방울과 함께 그리드를 헹 구 십시오. 이 단계는 초과 얼룩 솔루션을 제거 합니다.</li>
	<li>그리드 테이블에, 핀셋으로 눌러 놓고 실 온에서 10 분 동안 건조 문화 접시와 부분적으로 그리드를 커버 합니다.</li>
	<li>80 편으로 미래 관측을 위해 EM 격자 상자에 그리드를 저장 kV.</li>
</ol>
4. 전체 산 Immunostaining에 대 한
<ol>
	<li>글로우 방전 얇은 formvar/탄소 필름 코팅 200 메쉬 구리 EM 격자 30 s.</li>
	<li>2%의 1 mL와 함께 순화 exosomes 수정 Paraformaldehyde (PFA) 5 분. 주의: Paraformaldehyde 가스는 유독 합니다. 모든 작업은 통풍이 증기 두건에서 행해져야 한다.</li>
	<li>5-7 µ L exosome 솔루션에 고정 하 고 품 어 PBS의 100 µ L 5 분 린스에 대 한 3 시간 10 분 동안 각각.</li>
	<li>10 분 무료 알데하이드 그룹을 끄다 0.05 M 글리신의 50 µ L로 격자를 취급 합니다.</li>
	<li>30 분 동안 차단 버퍼 (PBS 포함 하는 1 %BSA)의 한 방울을 격자를 전송.</li>
	<li>1 시간 (필요한 경우,이 단계 실행 되어야 한다 4 ° C에서 하룻밤)에 대 한 반대로 PD L1 항 체 (0.1 %BSA 포함 하는 PBS에서 1: 100) 50-100 µ L로 격자를 품 어.</li>
	<li>10 분 동안 0.1 %BSA 포함 하는 PBS의 5 별도 방울 (50 µ L)과 그리드를 씻어.</li>
	<li>1 시간에 대 한 2차 항 체의 하락을 그리드를 전송 합니다.</li></ol>반대로 마우스 IgG 9-11 nm 금 입자에서 0.1 %BSA 포함 하는 PBS에서 1: 100 희석을 활용.
	<li>10 분 동안 0.1 %BSA 포함 하는 PBS의 5 별도 방울 (50 µ L)과 그리드를 씻어. DW의 두 별도 방울 (50 µ L)으로 그리드를 씻어. 3.8 3.4에서 설명한 대로 2 %uranyl 아세테이트와 부정적인 얼룩를 수행 합니다.</li>
	<li>80 편으로 미래 관측을 위해 EM 격자 상자에 그리드를 저장 kV.</li>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+biologic+characterization+of+exosomes+and+microvesicles+as+facilitators+of+HIV-1+infection+in+macrophages.">Biochemical and biologic characterization of exosomes and microvesicles as facilitators of HIV-1 infection in macrophages.</a> J Immunol. 189 (2), 744-754 (2012).</li><li>Gong, J., Korner, R., Gaitanos, L., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+mediate+cell+contact-independent+ephrin-Eph+signaling+during+axon+guidance.">Exosomes mediate cell contact-independent ephrin-Eph signaling during axon guidance.</a> J Cell Biol. 214 (1), 35-44 (2016).</li><li>Melo, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican-1+identifies+cancer+exosomes+and+detects+early+pancreatic+cancer.">Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.</a> Nature. 523 (7559), 177-182 (2015).</li><li>Valadi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nat Cell Biol. 9 (6), 654-659 (2007).</li><li>van Niel, G., Heyman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+cell+cytoskeleton+and+intracellular+trafficking.+II.+Intestinal+epithelial+cell+exosomes:+perspectives+on+their+structure+and+function.">The epithelial cell cytoskeleton and intracellular trafficking. II. Intestinal epithelial cell exosomes: perspectives on their structure and function.</a> Am J Physiol Gastrointest Liver Physiol. 283 (2), G251-G255 (2002).</li></ol>

저자는 공개 없다.

이 문서는 자세한 exosome의 구조를 관찰 하 고 그 특정 단백질의 라벨에 대 한 프로토콜을 표시 합니다. 부정적인 얼룩 exosome17이미징에 대 한 최선의 방법으로 간주 되었습니다. 이 기본 기술을 exosomes의 컵 모양의 구조를 보이고 있다. 그러나,이 컵 모양 인 위 건조 과정으로 인해 발생할 수 있는 형태입니다. 곳을 알아내는 가장 결과 exosomes 용액25,<sup clas...

Extracellular 소포 (EVs) 지질 bilayer 소포 세포에 의해 분 비 되며 그들의 크기 범위는 30 300 nm 사이. EVs는 Hodgkin의 질병1환자의 소포에서 증거와 함께 1978 년에 처음 보고 되었다. 이러한 vesicles 40 ~ 120 나노미터 크기에 보고 했다. 1980 년에, 그것은 EVs 응고 시스템 및 혈전 증, 암 환자2혈관 내 혈액 응고의 형성에 관련 된 것으로 알려졌다. 30 년 후, EVs 종양 침공, 면역 탈출 및 신생3홍보 하는 중요 한 요소 되도록 보고 되었습니다. 또한, EVs의 기능 세포의 상호 작용 분야에서 염증, 면역 장애, 신경 질환 및 암4레 귤 레이 터로 공부 되었다. EVs는 특정 생체 단백질, mRNA, 예측에 관한5등 포함, 진단 및 치료에 그들의 잠재적인 응용 프로그램 분석된6,7되었습니다. EVs는 exosomes, prostasomes, oncosomes, dexosomes, 미, promininosomes, argosomes, 및 그들의 세포질 근원 및 생물학 기능3에 따라 exosome 같은 소포를 포함 한 하위의 구성 범주. 또한, 그들의 속에 따라, 이러한 EVs 수 나눌 수 microvesicles (창 고 microvesicles, 100-1000 nm) 및 exosomes (30-300 nm)8,9. 이러한 가운데,는 exosome 면역 응답10, 암11,12, 및 전염병13셀 의사 소통으로 보고 되었습니다.
조기 진단 위한 바이오 마커도 exosomes에 대 한 관심, 고 정화와 exosomes의 분자 이미징 기술을 함께 동반 되어야 합니다. 다양 한 크기와 exosomes, 그들의 기원과 기능14, 따라의 형태학은 전자 현미경과 같은 높은 해상도와 현미경 기법에 의해 구분할 수 있습니다. 대부분 exosomes 부정적인 스테인드 전송 전자 현미경 (TEM)15,,1617에 의해 시각 했다 그리고이 결과 전체 산 소포에 특정 단백질의 immunolabeling에 의해 확인 되었다 18. 여러 연구 그룹 스캐닝 전자 현미경, 원자 힘 현미경19,20및 Cryo 편21,22를 통해 구조를 보고 있다. 그러나, 이러한 기술은 exosome 구조를 공부 하는 데 유용 하는 동안 그들은 충분 하지 않습니다는 exosome 안에 있는 특정 단백질의 위치를 관찰. 따라서, 우리는 특정 단백질의 라벨로 exosomes 이미징에 대 한 프로토콜 도입. 우리 블록 준비, ultrathin 단면화 및 immunostaining에 있는 exosome 단백질의 자세한 위치를 ascertaining에 대 한 적용. 이 부정적인 얼룩이 지 고는 exosome의 전통적으로 사용 되는 전체 산 immunostaining와 비교 되었다.

<table><tbody><tr><td>Glutaraldehyde</td><td>EMS</td><td>16200</td><td></td></tr><tr><td>Sodium cacodylate&amp;nbsp;</td><td>EMS</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide 1 g crystal</td><td>EMS</td><td>19100</td><td>Use only in fume hood</td></tr><tr><td>acetone</td><td>JUNSEI</td><td>1B2031</td><td></td></tr><tr><td>Spurr medium</td><td>TED PELLA</td><td>18108</td><td></td></tr><tr><td>Ultra-microtome</td><td>Leica</td><td>UCT</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400</td><td>Hazardous chemical</td></tr><tr><td>Lead citrate</td><td>EMS</td><td>17900</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>H7600</td><td></td></tr><tr><td>nickel grid</td><td>EMS</td><td>G200-Ni</td><td></td></tr><tr><td>Copper grid</td><td>EMS</td><td>G200-Cu</td><td></td></tr><tr><td>glycine</td><td>SIGMA</td><td>022K5404</td><td></td></tr><tr><td>Phosphate buffer saline</td><td>SIGMA</td><td>P4417</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Aurion</td><td>25557</td><td></td></tr><tr><td>1st Antibody</td><td></td><td></td><td>purification in manually&amp;nbsp;</td></tr><tr><td>Purified anti-human CD274 (B7-H1, PD-L1) Antibody</td><td>BioLegend</td><td>329710</td><td>Whole mount Immumogold</td></tr><tr><td>Purified Mouse IgG2b, &amp;kappa; Isotype Ctrl</td><td>BioLegend</td><td>401202</td><td>Whole mount Immumogold (Control)</td></tr><tr><td>2nd Anti-mouse igG conjugated 9-11 nm gold particle</td><td>sigma</td><td>G7652</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>JEOL</td><td>JEM2200FS</td><td></td></tr><tr><td>Glow discharger</td><td>JEOL</td><td>JFC1100E</td><td></td></tr><tr><td>Carbon grid</td><td>EMS</td><td>121119</td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>19210</td><td></td></tr><tr><td>Formvar carbon coated Copper Grid (200 meh)</td><td>EMS</td><td>FCF200-CU</td><td></td></tr><tr><td>Formvar carbon coated&amp;nbsp; Nickel Grid (200 mesh)</td><td>EMS</td><td>FCF200-NI</td><td></td></tr></tbody></table>

sample preparation and imaging of exosomes by transmission electron microscopy

Exosomes는 나노 크기의 세포 외 체액 분 비 소포 그리고 그들을 분 비 하는 셀의 특성을 나타내는 것으로 알려져 있습니다. 내용과 secreted vesicles의 형태 셀 행동 또는 생리 적 상태, 예를 들면 세포 성장, 마이그레이션, 분열, 그리고 죽음을 반영합니다. Exosomes의 역할 매우 크기에 따라 달라질 수 있습니다 따라 다르며 exosomes의 크기 30에서 300 nm. Exosome 이미징에 대 한 가장 널리 사용 되는 방법 부정적인 얼룩, Cryo-전송 전자 현미경, 스캐닝 전자 현미경, 원자 힘 현미경을 기반으로 하는 다른 결과 이다. 전형적인 exosome 형태학 부정적인 얼룩 통해 평가 컵 모양 이지만 더 세부 사항은 아직 명확. 구조 연구를 통해 잘 특징이 있는 exosome 의료 및 제약 분야에서 필요한 특히 이다. 따라서, 함수 종속 형태 라벨 exosome의 상세한 구조에서 특정 단백질 등 전자 현미경 기법에 의해 확인 되어야 한다. 자세한 구조를 관찰, ultrathin sectioned 이미지와 exosomes의 부정적인 스테인드 이미지 비교 되었다. 이 프로토콜에서 부정적인 얼룩이 지기, 전체 마운트 immuno 얼룩이, 블록 준비, 얇은 섹션, 그리고 면역-골드 라벨을 포함 하 여 exosomes의 영상에 대 한 전송 전자 현미경 검사 법 하는 것이 좋습니다.

현재, exosomes 전송 전자 현미경 검사 법에 의해 크기와 모양 범주로 분류 됩니다. 그림 2 는 부정적인 exosome 및 전체 마운트 상태에 면역 이라는 exosome 스테인드. 그림 3 얇은 단면 후 sectioned exosome와 면역 이라는 exosomes를 보여준다. 면역-골드 얼룩 특정 단백질의 항 체를 사용 하 여 긍정적으로 exosome 식별은 exosome 단백질의 종류를...

Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

샘플 준비 및 전송 전자 현미경 검사 법에 의해 Exosomes의 영상

이 프로토콜 전송 전자 현미경 검사 법이 부정적인 얼룩이 지기, 상세한 구조 및 면역-골드 라벨 exosomes에 특정 단백질의 위치를 확인 하려면 ultrathin 단면에 필요한 다양 한 기법을 설명 합니다.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

sample-preparation-imaging-exosomes-transmission-electron

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

38.7K 조회수.  Asan Medical Center. 이 프로토콜 전송 전자 현미경 검사 법이 부정적인 얼룩이 지기, 상세한 구조 및 면역-골드 라벨 exosomes에 특정 단백질의 위치를 확인 하려면 ultrathin 단면에 필요한 다양 한 기법을 설명 합니다.

비디오: Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

원자력 현미경 검사법에 의한 세포 외 소포의 화상 진찰

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

연구

생화학

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

랜덤 코니컬 틸트 메서드를 사용하여 Exosome 단지의 단일 입자의 전자 현미경의 재건

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

생물학

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

종양 유래 엑 소 솜을 정량화 하는 나노 플라즈마-강화 된 산란 및 저 배율 현미경 이미징 사용

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

생체공학

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

격리 및 프로파일 링 마이크로 RNA-포함하는 인간의 담즙에서 엑소 좀을

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

의학

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA 함유 Exosomes의 분리 및 특성

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

엑소 정량화 및 크기 측정을위한 혁신적인 방법

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

생물의학 응용 분야에서 엑소좀의 지속적인 전달을 위한 마이크로니들 패치

Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging from drug delivery and regenerative medicine to disease diagnosis and tumor immunotherapy. However, the rapid clearance by traditional bolus injection and poor stability of exosomes restrict their clinical application. Microneedles serve as a solution that prolongs the residence time of exosomes at the administration site, thereby maintaining the drug concentration and facilitating sustained therapeutic effects. In addition, microneedles also possess the ability to maintain the stability of bioactive substances. Therefore, we introduce a microneedle patch for loading and delivering exosomes and share the methods, including isolation of exosomes, fabrication, and characterization of exosome-loaded microneedle patches. The microneedle patches were fabricated using trehalose and hyaluronic acid as the tip materials and polyvinylpyrrolidone as the backing material through a two-step casting method. The microneedles demonstrated robust mechanical strength, with tips able to withstand 2 N. Pig skin was used to simulate human skin, and the tips of microneedles completely melted within 60 s after skin puncture. The exosomes released from the microneedles exhibited morphology, particle size, marker proteins, and biological functions comparable to those of fresh exosomes, enabling dendritic cells uptake and promoting their maturation.

저자 스포트라이트: Innovative Microneedle-Based Strategies for Enhanced Exosome Delivery and Stability

Exosomes possess significant clinical potential, but their practical application is limited due to easy in vivo clearance and poor stability. Microneedles present a solution by enabling localized delivery by puncturing physiological barriers and dry-state preservation, thereby addressing the limitations of exosome administration and expanding their clinical utility.

엑소좀의 로딩 및 전달을 위한 Microneedle patches의 제작 및 특성 분석

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

3D 생물반응기 배양에 의한 활액 중간엽 줄기세포 유래 엑소좀의 대규모 제조

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

샘플 준비 및 전송 전자 현미경 검사 법에 의해 Exosomes의 영상

요약

더 많은 비디오 탐색

이 비디오의 챕터

원자력 현미경 검사법에 의한 세포 외 소포의 화상 진찰

랜덤 코니컬 틸트 메서드를 사용하여 Exosome 단지의 단일 입자의 전자 현미경의 재건

종양 유래 엑 소 솜을 정량화 하는 나노 플라즈마-강화 된 산란 및 저 배율 현미경 이미징 사용

격리 및 프로파일 링 마이크로 RNA-포함하는 인간의 담즙에서 엑소 좀을

RNA 함유 Exosomes의 분리 및 특성

엑소 정량화 및 크기 측정을위한 혁신적인 방법

엑소좀의 로딩 및 전달을 위한 Microneedle patches의 제작 및 특성 분석

엑소좀의 로딩 및 전달을 위한 Microneedle patches의 제작 및 특성 분석

엑소좀의 로딩 및 전달을 위한 Microneedle patches의 제작 및 특성 분석

3D 생물반응기 배양에 의한 활액 중간엽 줄기세포 유래 엑소좀의 대규모 제조