Name: isolation and characterization of extracellular vesicles from adult schistosoma japonicum
Uploaded: 2018-05-22T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

이 연구는, 일부 또는 전체, 국가 자연 과학 재단의 중국 (31472187 및 31672550) 및 농업 과학, 농업 과학의 중국 아카데미의 기술 혁신 프로그램 지원.

모든 동물 실험 상하이 수의학 연구소, 농업 과학의 중국 아카데미의 로컬 윤리 위원회에 의해 승인 (허가 번호: SHVRIAU-14-0101).
1. Schistosomes 의 생체 외에서 문화
참고: Schistosomes 는 생물을 나타냅니다. 노동자 때 생물 학적 재료 관련 웜, schistosomal 정지, 또는 다른 처리 시간 라텍스 장갑을 전혀 착용 해야 한다. 뉴질랜드 토끼 ...

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EVs에 대 한 최근 연구는 schistosome EVs 호스트 병원 체 상호 작용3,9,,1216에 중요 한 역할을 설명 했다. 추가 주소에 그들의 규정 하는 기능 필수적 이다 schistosomes에서 EVs를 분리. 여기, 우리 SjEV 절연을 위한 다른 방법을 설명합니다. 이 메서드 생성, SjEVs의 넓은 범위 크기 100에서 400 nm nm, 성인 미 나무</...

Extracellular 소포 (EVs)는 작은 막 도약 소포와 다양 한 단백질, 지질, 핵 산 캡슐화의 인구. 최근 연구 보여준 EVs 셀 셀 통신에 중요 한 역할을 하 고 셀 개발, 면역 조절, 신생, 세포 마이그레이션2, 등 수많은 생리 적 프로세스의 규칙에 관련 된 3,,45. 증거를 축적 EVs, exosomes, 그리고 그들의 miRNA 화물 순환 특정 질병6의 잠재적인 생체 나타냅니다 나타냅니다.
여러 가지 원생 동물 Trichomonas vaginalis, Trypanosoma cruzi, 등 Leishmania spp., EVs;를 분 비 수 표시 되었습니다 helminths 발견 되었습니다 또한7호스트 생활에 EVs를 분 비. 기생 EVs pathogenicity8, 감염과 면역 규칙9의 유지 보수에 포함 될 표시 되었습니다. 최근 두 Schistosoma mansoni (S. mansoni) 10,11 schistosomes에서 공부와 미 나무12 성인 schistosomes 분 수 있는 exosome 같은 소포 비 표명 특정 생물 학적 과정의 기능 규정에 참여.
날짜 하려면, 여러 가지 방법은 extracellular 소포, ultracentrifugation, 외13, 폴리머 기반 시 약, 크기 배제 크로마토그래피, immunoaffinity 격리의 사용을 사용 되었습니다. 이러한 다른 방법을 그들의 자신의 이점 및 제한 사항14. 일반적으로, ultracentrifugation는 소포 격리에 대 한 표준 방법으로 간주 됩니다. 그러나,이 방법은 잠재적인 EV 집계14의 한계에서 고통.
Schistosomes에서 방법의 몇 가지 EV 격리에 대 한 보고 되었습니다: ultracentrifugation11,,1210 와 여러 단계 (달걀16, 상업 EV 격리 키트16 를 사용 하 여 포함 됩니다 schistosomula11, 성인 schistosomes10,,1217). Microvesicles 천 나노미터 몇 백 나노미터 크기의 넓은 범위는, 그 주어진 우리 벤치 원심 분리기, 한, 그리고 분리에서 EVs 상업 킷은 절연을 사용 하 여 결합 하는 대체 방법 개발 성인 Schistosoma 나무. 격리 된 EVs는 일반적으로 100-400 nm의 직경을 소유 하 고 성공적으로 받는 세포에 의해 내 면화 했다.

<table border="0" cellpadding="0" cellspacing="0" style="width:1161px;" width="1160">
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	</colgroup>
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:459px;">Material</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:459px;">Total Exosome Isolation Reagent (from cell culture media)</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td style="width:188px;">4478359</td>
			<td style="width:319px;">For isolating S. japonicum extracellular vesicles</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">PKH67</td>
			<td style="width:196px;">Sigma-aldrich</td>
			<td style="width:188px;">MINI67</td>
			<td style="width:319px;">For labeling Evs</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">RPMI 1640 Medium</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>11875119</td>
			<td>For parasite culture</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Penicillin-Streptomycin</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">0.22&#956;m syringe filter</td>
			<td style="width:196px;">Merck</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">SnakeSkin Dialysis Tubing</td>
			<td style="width:196px;">Thermo SCIENTIFIC</td>
			<td>68035</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">PEG8000</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>A100159&#160;</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">RIPA</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0013B</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">EMD Millipore&#8482; Immobilon&#8482; Western Chemiluminescent HRP Substrate (ECL)</td>
			<td style="width:196px;">Fisher scientific</td>
			<td>WBKLS0100&#160;</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">DAPI</td>
			<td>Cell Signaling Technology</td>
			<td>4083</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">BCA kit</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0010</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">CD63 antibodies</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>D160973-0025</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">PVDF</td>
			<td>Bio-Rad</td>
			<td>1704273</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Formvar/Carbon 400 mesh</td>
			<td>Ted Pella</td>
			<td>01754-F</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Phosphotungstic Acid</td>
			<td>Ted Pella</td>
			<td>19402</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">anti-mouse IgG-HRP</td>
			<td>CWBIO</td>
			<td>CW0102</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">NCTC clone 1469 cells</td>
			<td style="width:196px;">ATCC</td>
			<td style="width:188px;">ATCC&#174; CCL-9.1&#8482;</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">FBS</td>
			<td style="width:196px;">HyClone</td>
			<td style="width:188px;">SV30087.02</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:459px;">Equipments</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">GE chemoluminescance imaging system</td>
			<td>GE</td>
			<td>ImageQuant LAS4000mini</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Transmission electron microscopy</td>
			<td>Hitachi</td>
			<td>H-7600</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Milli-Q water</td>
			<td>Milli-Q</td>
			<td>Milli-Q Elix</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Eppendor Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5804R</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Zetasizer Nano</td>
			<td>Malvern</td>
			<td>Zetasizer Nano ZS&#160;</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Ultracentrifuge</td>
			<td style="width:196px;">Beckman</td>
			<td colspan="2">Optima L-100 XP Ultracentrifuge</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Incubator</td>
			<td style="width:196px;">ESCO</td>
			<td>CCL-107B</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Microscope</td>
			<td style="width:196px;">Zeiss</td>
			<td>Zeiss Axin Observer Z1</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

Extracellular 소포 (EVs)는 막 소포 세포 외 microenvironment로 셀의 다양 한 발표. EVs는 누구의 크기 범위 40 1000 nm 사이 이기종 vesicles의 인구를 나타냅니다. 축적 된 증거는 EVs에 중요 한 역할 규정 호스트 병원 체 상호 작용에 표시. Schistosome EVs에 대 한 깊은 이해는 기본 schistosomiasis에 대 한 새로운 전략의 개발을 활성화 하는 schistosome-호스트 상호 작용 메커니즘에 대 한 통찰력을 제공 해야 합니다. 여기, 우리는 성인 Schistosoma 나무 (S. 머 위)에서 절연 및 EVs의 특성화에 대 한 프로토콜을 제시 하 여 EVs 함수 schistosomes에 진학 하고자 합니다. EVs 문화 매체 체 외에 상업 exosome 격리 키트와 결합 하는 원심 분리를 사용 하 여 격리 했다. 고립 된 미 나무 EVs (SjEVs) 일반적으로 100-400 nm의 직경을가지고 그리고 전송 전자 현미경 검사 법 및 서쪽 blotting에 의해 특징입니다. 염료-표시 된 SjEVs PKH67의 사용 SjEVs 받는 사람 세포에 의해 내 면화는 설명 했다. 전반적으로, 우리의 프로토콜 성인 schistosomes;에서 EVs를 격리 하기 위한 대체 방법을 제공 한다 격리 된 SjEVs 기능 분석에 적합 수 있습니다.

설명 된 프로토콜을 사용 하 여 격리 된 SjEVs의 수익률을 계량, 우리는 28 일 성인 schistosomes에서 고립 된 SjEVs의 단백질 농도 액세스할 수 BCA 단백질 분석 결과 사용. 표 1에서 같이, SjEV 단백질 농도의 매체 (표 1) 100 mL 당 250 µ g을 208 µ g에서 배열 했다. 입자 크기 Malvern 나노 분석에 의해 결정 된 대로 표시 된 격리 SjEVs 100에서 240 400 nm nm, 높은 인구 약...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

절연 및 Extracellular 소포 특성 성인 Schistosoma 나무 에서

여기, 선물이 성인 Schistosoma 나무에서 체 외에서 배양된 매체에 extracellular 소포 (EVs) 격리를 설명 하는 프로토콜.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

The overall goal of this procedure is to isolate extracellular vesicles from the medium of in vitro cultured adult Schistosoma japonicum. This method can efficiently isolate EV in in vitro culture medium from adult Schistosoma japonicum. The main advantage of this technique is able to efficiently isolate extracellular vesicle from short-term in vitro culture medium, which can minimize the stress potentially result in non-physiological EV secretion.Demonstration will be by Juntao Liu, a technician from my lab. Collect the parasites in a 500-milliliter beaker, and wash them thoroughly but gently three times using 200 milliliters of warm PBS. Keep the washes brief, and use gravity sedimentation to separate the parasites.Next, onto 90-millimeter dishes, plate about 200 worm pairs per dish. In the dish, wash the parasites twice more using gravity sedimentation. For these washes, use warm RPMI medium supplemented with pen-strep.Replace the second wash with 40 milliliters of the medium without antibiotics. Then, culture the parasites at 37 degrees Celsius under 5%carbon dioxide for two hours. After two hours, transfer the media from the plates to centrifuge tubes, and then pool the eggs and worm debris at 2, 000 g for 30 minutes at four degrees Celsius.Then, collect and transfer the supernatant to a fresh tube. Next, filter the supernatant through a 0.22-micron syringe filter, and collect the filtered solution into a dialyzing bag with a molecular weight cut-off of 3.5 kilodaltons. Then, dialyze the solution using 8%PEG8000 solution at four degrees Celsius overnight.This step is important for increasing the yield of extracellular vesicles. The next day, transfer the dialyzed solution to a new tube, and add 0.5 volumes of the total exosome isolation reagent. Mix the solution until it is homogeneous, and incubate at about five degrees Celsius overnight.The following morning, centrifuge the mixture at 15, 000 g for one hour at about five degrees Celsius. Next, aspirate and discard the supernatant, and resuspend the pellet of EVs in two milliliters of PBS. The pellet will not likely be visible.For western blot analysis, first determine the protein concentration of the sample, such as with the BCA assay. Then, prepare 10 to 20 micrograms of EVs in 22.5 microliters of RIPA buffer. Next, add 7.5 microliters of 4X loading buffer, and heat the mixture at 95 degrees Celsius for five minutes.After the heat treatment, separate the extracellular vesicle proteins on a 12%SDS-PAGE gel. After running the gel, the proteins are transferred into PVDF membrane. Probe the membrane with anti-CD63 antibodies at a one to 1, 000 dilution.Then, treat the membrane with anti-rabbit IgG-HRP at a one to 5, 000 dilution. Now, use an ECL detection reagent to develop the membrane, and follow with analysis on a chemiluminescence imaging system. Start with transferring five micrograms of sample into a polypropylene ultracentrifuge tube, and adjust the volume to 12 milliliters with RPMI-1640 medium.Then, centrifuge the sample at 110, 000 g for 90 minutes at four degrees Celsius. After the centrifugation, carefully aspirate and discard the supernatant. Then, prepare a 2X extracellular vesicle suspension by adding one milliliter of diluent C to the pellet.Resuspend the pellet with gentle pipetting. Now, prepare fresh 2X dye solution. Add two microliters of the PKH67 ethanolic dye to one milliliter of diluent C in a microfuge tube, and mix well.Then, stain the sample by mixing together the two solutions and incubating the mix at room temperature for four minutes. Next, stop the staining by adding four milliliters of FBS and waiting another minute. Then, add RPMI-1640 medium to adjust the volume to 12 milliliters, and spin down the suspension to isolate the stained vesicles.Remove and discard the supernatant. Then, resuspend the pelleted PKH67-labeled EVs in 12 milliliters of RPMI-1640 medium. Then, repeat the centrifugation, but this time collect the vesicles in one milliliter of PBS.For the uptake assay, prepare overnight cultures of mammalian cells in six-well plates. The next morning, add five micrograms of PKH67-labeled vesicles to each well, and continue the culture for two to four hours. After the incubation, remove the medium, and wash the cells with PBS two times.Then, fix the cells for 15 minutes, wash them twice with PBS, and stain them with DAPI before imaging. Schistosomes at the liver stage were collected from rabbits 28 days post-infection. Extracellular vesicles were then collected from the schistosomes worms and analyzed.Their particle size, as determined by Malvern nanoparticle analysis, ranged between 100 and 400 nanometers, with the bulk of the population near 220 nanometers. Transmission electron microscopy revealed the extracellular vesicles to be round and between 100 and 200 nanometers in diameter. Western blotting analysis indicated that the isolated extracellular vesicles were recognized using CD63 antibodies, which is a typical extracellular vesicle marker.Fluorescence microscopy of PKH67-labeled extracellular vesicles internalized by the NCTC clone 1469 cells showed that the extracellular vesicles were mainly distributed in the cytoplasm of recipient cells. After watching this video, you should have a good understanding of how to isolate EV in in vitro culture medium from adult Schistosoma japonicum.

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

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