이 작품은 캘리포니아 대학, 리버 사이드와 미국 농 무부 식품 및 농업, 해치 프로젝트 1009710 J.P.G.에 의해 지원 되었다 이 자료는 J.P.G.에 보조금 번호 1817363에서 국립 과학 재단에서 지 원하는 작업 기반

1. A. thaliana 식물 성장
<ol><li>5 cm x 5 cm 일회용 냄비에 뿌리 다 A. thaliana 씨앗 표준 토양 혼합 가득. 32 이러한 냄비의 물으로 채워진 플라스틱 쟁반에 넣어 (~ 0.5 c m 깊이)는 식물을 가진 플라스틱 트레이 식물 성장 챔버로 전송.<ol>
<li>다음과 같이 성장 챔버 설정을 설정: 200 µ / ms 광합성 활성 방사선 (파), 24 ± 1 ° C 일 및 21 ± 1 ° C 밤, 습도 60%, 및 14/10 h 주/야간 라이트 정권, 각각.</li>
	</ol>
</li>
	<li>발 아의 1 주 후 하나의 개별 공장을 두고 각 냄비 얇은. 각 냄비에서 비슷한 크기와 묘를 계속 기록해 둡니다.</li>
	<li>물이 냄비 플라스틱 쟁반에 직접 붓는 수도 물으로 한 번 매 2 일. 4 주에 대 한 식물을 성장. A. thaliana 식물 추가 사용 하기 위해 준비가 되었습니다.</li>
</ol>2. 합성 및 PNC의 특성화
<ol><li>세 륨 (III) 질산염의 1.08 g 무게 그리고 분자 생물학 학년 물 50 mL 원뿔 튜브에서 2.5 mL에 용 해.</li>
	<li>50 mL 원뿔 튜브에 분자 생물학 학년 물 5 mL에 용 해 및 폴 리 (아크릴) 산의 4.5 g 무게.</li>
	<li>디지털 소용돌이 믹서를 사용 하 여 15 분 동안 2000 rpm에서 철저 하 게 이러한 두 가지 솔루션을 혼합.</li>
	<li>수산화 암모늄 솔루션 (7.2 M)의 15 mL 50 mL 유리 비 커에 전송.</li>
	<li>500 rpm에서 교 반 하면서 dropwise 수산화 암모늄 솔루션에 혼합 단계 2.3에서 추가 하 고 증기 두건에서 24 시간 동안 실 온에서 500 rpm에서 저 어.</li>
	<li>동안을 피하기 위해 솔루션의 상당한 손실 하룻밤 반응 종이의 조각으로 비 커를 커버.</li>
	<li>24 시간 후 결과 솔루션 50 mL 원뿔 튜브에 전송 하 고 모든 가능한 파편 및 큰 응집 체를 제거 하는 1 h 3900 x g에서 원심.</li>
	<li>표면에 뜨는 솔루션의이 22.5 mL 3 15 mL 10 kDa 필터에 전송 하 고 분자 학년 물 45 mL의 총 희석 수 있도록 필터의 나머지를 입력 합니다.</li>
	<li>무료 고분자에서 표면에 뜨는 솔루션을 정화 하 고 15 mL 10 kDa 필터와 15 분에 3900 x g에서 원심 분리기에는 상쾌한을 추가 하 여 벤치탑 원심 분리기와 다른 시 약 6 번 이상이 단계를 반복.</li>
	<li>아니 무료 고분자를 위해 220-700 nm에서 대 일 분 광 광도 계를 각 주기에서는 eluent의 흡 광도 측정 하 고 다른 시 약은 최종 PNC 솔루션에 존재.</li>
	<li>5 mL 주사기로 수집한 PNC 솔루션을 20 nm 기 공 크기 주사기 필터에 대 한 필터링. 필터링 된 PNC 솔루션 50 mL 원뿔 튜브에 수집 합니다.</li>
	<li>플라스틱 베트에 희석된 최종 PNC 솔루션을 대 일 분 광 광도 계 220-700 nm에서의 흡 광도 측정. PNC 흡 광도 피크는 271 nm.</li>
	<li>맥주 Lambert 법률을 사용 하 여 그것의 농도 계산: A = εCL. A는 주어진된 샘플에 대 한 피크 값의 흡 광도, ɛ PNC (c m-1 M-1)의 어 금 니 흡수 계수, L 광학 경로 길이 (베트 폭,이 방법에서는 1 cm) 이며 C는 측정 된 나노 입자의 어 금 니 농도.</li>
	<li>유체역학 직경 및 분석기를 사용 하는 입자 크기와 제타 잠재적인 (그림 1) 합성된 PNC의 제타 전위를 측정 합니다.</li>
	<li>추가 사용까지 냉장고 (4 ° C)에서 최종 PNC 솔루션을 저장 합니다. 참고: 우 외를 참조 하십시오. PNC 특성화에 대 한 더 많은 프로토콜 세부 정보에 대 한 8 .</li>
</ol>3. 라벨 DiI 형광 염료와 PNC
<ol><li>학년 물 20 mL 유리 유리병에 분자 생물학의 3.6 mL와 5 m m (58 mg/L) PNC의 0.4 mL를 혼합 하 고 500 rpm에서 저 어.</li>
	<li>24 µ L 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine 과염소산염 염료 솔루션 추가 (DiI, 2.5 mg/mL; DMSO에 희석) DiI 염료 솔루션 DMSO (디 메 틸 sulfoxide)의 176 µ L로.</li>
	<li>PNC 솔루션, 주위 온도에 1 분 동안 1000 rpm에서 교 반에 dropwise DiI 염료를 추가 합니다.</li>
	<li>15 mL 10 kDa 필터에이 결과 혼합물을 전송 하 고 분자 생물학 학년 물 총 희석을 가기로 튜브를 채울 15 mL.</li>
	<li>5 분 동안 3900 x g에서 15 mL 10 kDa 필터와 DMSO 및 모든 가능한 무료 DiI 염료에서 PNC (DiI-PNC) 솔루션 벤치탑 원심 분리 하 여 표시 하는 DiI를 정화.<ol>
<li>단계 3.5 5 번 이상 반복 합니다.</li>
	</ol>
</li>
	<li>20 nm 기 공 크기 주사기 필터를 통해 최종 DiI PNC 솔루션을 필터링 합니다.</li>
	<li>대 일 분 광 광도 법에 의해 최종 DiI-PNC의 흡 광도 측정 하 고 맥주 Lambert 법률 (그림 2)의 농도 계산. 자세한 내용은 단계 2.13를 참조 하십시오.</li>
	<li>추가 사용에 대 한 4 ° C에서 냉장고에 보관.</li>
</ol>4. 식물의 침투 PNC와 나뭇잎
<ol><li>침투의 0.1 mL를 추가 그것은 0.5 m m PNC 또는 DiI PNC 솔루션 및 소용돌이의 0.9 mL에 (100 mM TES, 100 m m MgCl2, pH 7.5, HCl에 의해 조정)를 버퍼. 부정적인 통제로 10mm TES 침투 버퍼의 솔루션을 사용 합니다.</li>
	<li>1 mL 균 needleless 주사 통에 PNC 또는 DiI PNC 침투 솔루션의 0.2 mL를 전송. 가능한 모든 기포를 제거 하려면 누릅니다.</li>
	<li>식물 성장 챔버 룸 라이트 조건에서 가능한 stomata 폐쇄를 피하기 위해 나노 입자와 침투 직전에서 검색 합니다.</li>
	<li>침투 하기 전에 엽록소 측정기를 사용 하 여 비슷한 크기와 A. thaliana 잎에서 엽록소 콘텐츠 측정. 측정 3 복제와 각 리프 (3 개 이상 측정의 구성 된 각 복제)14. A. thaliana 나뭇잎 침투 실험에 대 한 비슷한 엽록소 콘텐츠를 선택 합니다.</li>
	<li>부드럽게 리프 lamina (abaxial 쪽)의 하단에 대 한 needleless 주사기의 끝을 누르면 천천히 최근 준비 PNC 또는 DiI PNC 솔루션으로 잎에 침투 하 고 플런저 (그림 3A)를 누릅니다.</li>
	<li>부드럽게 닦아 내는 섬세 한 작업 퍼그림 3(C)를 사용 하 여 리프 lamina (그림 3B)의 표면에 남아 있는 과잉 솔루션 및 공장 레이블. 새로운 섬세 한 작업 잎사귀를 사용 하 여 잎의 각 그룹에 대 한.</li>
	<li>계속 침투 A. thaliana 식물 잎 적응 및 3 h에 대 한 PNC와 DiI PNC 보육에 대 한 벤치에. 참고: 침투 A. thaliana 식물은 더 사용 (그림 3D)에 대 한 준비 다음.</li>
</ol>5. Confocal 현미경 검사 법에 대 한 리프 샘플의 준비
<ol><li>롤은 완두콩 크기의 관찰 1 ㎝ 반경(그림 4)에 대 한 하 젤 양과 다음 1mm 얇은 유리 슬라이드 (그림 4B) 될 때까지 밖으로 확산.</li>
	<li>코르크 송곳을 사용 하 여 (직경 0.3 c m) 유리 슬라이드 (그림 4C)에 관찰 젤의 중심에 원형 부분을 잘라.</li>
	<li>채워 컷 잘 완전히 perfluorodecalin (PFD) 잎 조직에 깊이 및 더 나은 confocal 영상 확인.</li>
	<li>코르크 송곳을 사용 하 여 (직경 0.2 c m) 적응된 DiI PNC에서 잎 디스크 수집 하 침투 A. thaliana 식물 (그림 4D).</li>
	<li>가득 PFD에 리프 디스크 탑재 잎의 침투 (abaxial) 쪽 얼굴.</li>
	<li>잎 디스크 위에 사각형 coverslip 넣고 조심 스럽게 관찰 젤 잘 밀봉 하 고 아무 공기 방울 갇혀 (그림 4E) 유지를 균등 하 게 슬라이드 coverslip에 누릅니다.</li>
</ol>6. 이미징 DiI-PNC Confocal 현미경 검사 법에 의해 잎 조직에
<ol><li>40 X 객관적 렌즈는 거꾸로 레이저 confocal 현미경 검사에 사용 합니다.</li>
	<li>2 3 드랍 스 ddH2O 40 X 객관적 렌즈 상단에 드롭.</li>
	<li>객관적인 렌즈 거꾸로 40 위에 준비 된 DiI PNC 침투 잎 샘플 슬라이드를 놓습니다.<ol>
<li>Coverslip 편이지만 렌즈에 ddH2O와 직접 접촉을 슬라이드 유리 하지 있는지 확인 하십시오.</li>
	</ol>
</li>
	<li>레이저 빛 또는 밝은 분야 현미경 샘플에 관심 영역을 찾아.</li>
	<li>현미경 소프트웨어를 시작 하 고 (20% 설정) 아르곤 레이저를 켭니다.</li>
	<li>광학 슬라이스 2 µ m 및 4 선 평균 미만 수집 pinhole을 설정 합니다.</li>
	<li>Confocal 현미경 설정을 샘플 이미지: 514 nm 레이저 여기 (30%); Z-스택 섹션 두께: 2 µ m; PMT1: 550-615 nm (대 한 DiI PNC 영상); PMT2: 700-800 nm (엽록체 영상)에 대 한.</li>
	<li>다른 개인에서 리프 샘플의 대표적인 confocal 이미지, 3 생물의 최소 복제.</li>
</ol>7. 이미징 PNC에서 vivo에서 Confocal 현미경 검사 법에 의해 청소 선생님
<ol><li>준비 25 µ M 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA, 일반 선생님을 나타내는 염료)와 10 µ M dihydroethidium (DHE, superoxide 음이온을 나타내는 염료) 1.5 mL microcentrifuge 튜브에서 TES 침투 버퍼 (pH 7.5)에 염료 별도로.</li>
	<li>코르크 송곳을 사용 하 여 (직경 0.2 cm) 잎을 수집 하는 적응된 PNC에서 디스크 A. thaliana 식물 침투.<ol>
<li>염료 로드 프로세스를 가속 화 하기 위해 잎 디스크에 3 ~ 4 구멍을는 집게의 날카로운 팁을 사용 합니다.</li>
	</ol>
</li>
	<li>이동 잎 디스크 microcentrifuge 튜브 H2DCFDA 및 DHE 별도로 하 고 어둠 속에서 30 분 동안 품 어.</li>
	<li>부 화, 후 린스 잎 ddH2O 3 번 디스크와 유리에 관찰 슬라이드 마운트 (프로토콜 섹션 5를 참조) 젤.</li>
	<li>Confocal 현미경에 슬라이드를 넣고 수동으로 리프 mesophyll 셀의 지역에 초점. 자세한 내용은 프로토콜 섹션 6을 참조 하십시오.</li>
	<li>UV-a 잎 디스크 노출 (405 nm) 레이저 3 분 선생님을 생성 하 고 시계열 ("xyt") 잎 디스크 당에 선생님 신호 강도 변화를 기록.</li>
	<li>Confocal 현미경 설정으로 리프 디스크 이미지: 40 X 물 목표; 496 nm 레이저 여기; PMT1: 500-600 nm (DHE 및 DCFDA 염료 검출);에 대 한 PMT2: 700-800 nm (엽록체 감지)에 대 한. 부정적인 컨트롤로 침투 버퍼 솔루션에 침투 하는 식물을 사용 합니다.</li>
</ol>8. PNC 청소의 H2O2체 외
<ol><li>이전 간행물3,,815 에 방법에 따라 합성 PNC에는 생체 외에서 의 고양이 (catalase) 모방 활동을 수행
</li>
	<li>45.4 추가 1 x TES 침투의 µ L 버퍼 (10 m m TES, 10 m m MgCl2, pH 7.5, HCl에 의해 조정), 피츠버그 (60 nM, 3 µ L), 및 H2O2 (2 µ M, 1 µ L) 잘 (흰색 둥근 바닥 96 잘 접시)에 pipetting으로 부드럽게 섞는다.</li>
	<li>추가 10-아 세 틸-3, 7-dihydroxyphenoxazine (작업 농도 100 µ M, 0.5 µ L) 및 양 고추냉이 과산화 효소 (HRP; 작업 농도 0.2 U/mL, 0.1 µ L) 우물에 부드럽게 pipetting, 그것을 혼합 하 고 30 분 10-아 세 틸-3, 7-dihydroxyphenoxazine에 대 한 그것을 품 어 H2O2 와 반응 하 고 HRP의 resorufin로 변환 됩니다.<ol>
<li>랩는 인큐베이션 기간 동안 빛을 피하기 위해 알루미늄 호 일로 접시.</li>
		<li>H2O2를 대체 하기 위하여 물 또는 반응 버퍼를 사용 하 여 부정적인 제어를 준비 합니다.</li>
		<li>재고 솔루션을 제외한 주위 온도에 다른 모든 솔루션을 준비 합니다.</li>
	</ol>
</li>
	<li>플레이트 리더를 부 화 후 560에서 흡 광도 모니터링 nm resorufin를 사용 하 여 H2O2의 수준을 나타냅니다. 0, 2, 5, 10, 20, 및 30 분에서 시간 체제를 설정 합니다.</li>
</ol>

<ol>
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저자는 공개 없다.

이 프로토콜에 우리 PNC 합성, 특성, 형광 염료 라벨, 및 그들의 비보에 선생님 청소 활동을 전시 식물 mesophyll 세포 내에서 나노 입자의 confocal 영상 설명 합니다. PNC는 세 륨 질산염 및 PAA 솔루션에서 수산화 암모늄의 혼합물에서 합성 됩니다. PNC 흡수 spectrophotomery와 맥주 Lamberts 법을 사용 하 여 결정 하는 농도 의해 특징. 제타 잠재적인 측정 엽록체8납품을 강화 하기 위한 PN...

세 륨 산화물 나노 입자 (nanoceria) 생물 공학 능력1,,23청소의 독특한 촉매 반응성 산소 종 (선생님) 때문에 기초 연구에서 널리 사용 됩니다. Nanoceria는 두 산화 상태 (Ce3 + 및 Ce4 +) 4,,56사이 대체 표면 산소 공석의 큰 숫자 때문에 능력을 청소 하는 선생님. Ce3 + 매달려 유대는 nanoscale에 격자 긴장 redox 반응7자전거를 통해 이러한 결함 사이트의 재생을 촉진 하는 동안 선생님을 효과적으로 청소. Nanoceria는 또한 최근 사용 되었습니다 공부 하 고 엔지니어링 공장 기능8,9. Abiotic 스트레스 식물 선생님, 지질, 단백질 및 DNA10산화 손상의 축적 경험. A. thaliana 식물, 높은 빛, 열, 및 재미 스트레스8에서 향상 된 식물 광합성에 리드 nanoceria 촉매 vivo에서 선생님의 청소. 토양도 밀 (Triticum aestivum)11;의 증가 촬영 바이오 매스 및 곡물 수율 적용 nanoceria nanoceria로 치료 하는 canola (브라 시카 napus) 식물 소금 스트레스12에서 더 높은 식물 바이오 매스에 있다.
Nanoceria는 bioengineers를 제공 하 고 식물 생물학 abiotic 스트레스 응답을 이해 하 고 식물 abiotic 스트레스 내성을 강화 하는 나노기술 기반 도구. 선생님 청소 기능 vivo에서 Nanoceria의 식물 종의 독립적인 있으며 식물 조직에 손쉬운 배달 모형 유기 체 이상으로 광범위 한 응용 프로그램을 사용 하도록 가능성이 있다. 다른 유전자 기반 메서드와 달리 nanoceria 청소 능력13높은 선생님에 대 한 항 산화 효소의 overexpression 공장 라인을 생성 필요 하지 않습니다. Nanoceria 식물의 잎 lamina 침투 연구소 기반 연구에 대 한 실용적인 접근 방법입니다.
이 프로토콜의 전체 목표 1) 합성 부정 청구 폴 리 (아크릴) 산 nanoceria (PNC)의 특성화, 2) 납품 및 리프 셀에 걸쳐 PNC의 추적 및 3)의 모니터링 PNC 기반 선생님 청소 에서 설명 하는 vivo. 이 프로토콜에서 부정 청구 폴 리 (아크릴) 산 nanoceria (PNC)는 합성 하 고 그들의 흡수 스펙트럼, 유체역학 직경, 및 잠재적인 제타 특징. 식물으로 잎 조직 PNC를 제공 하는 간단한 잎 lamina 침투 방법을 설명 합니다. 이미징에 대 한 vivo에서 mesophyll 셀 내에서 나노 분산의, 형광 염료 (DiI) 레이블을 PNC (DiI-PNC)과 공초점 형광 현미경 검사 법을 통해 나노 입자 관찰에 사용 되었다. 마지막으로, 우리는 vivo에서 confocal 현미경 검사 법을 통해 PNC 선생님 청소 모니터링 하는 방법을 설명 합니다.

<table><tbody><tr><td>Cerium (III) nitrate hexahydrate</td><td>Sigma-Aldrich</td><td>238538-100G</td><td></td></tr><tr><td>Molecular Biology Grade Water, Corning</td><td>VWR</td><td>45001-044&amp;nbsp;</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>VWR</td><td>14-959-49A</td><td></td></tr><tr><td>Poly (acrylic acid) 1,800 Mw</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer</td><td>Fisher Scientific</td><td>02-215-391</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer Accessories: Foam Insert Set</td><td>Fisher Scientific</td><td>02-215-395</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma-Aldrich</td><td>05002-1L</td><td></td></tr><tr><td>PYREX Griffin Beakers, Graduated, Corning</td><td>VWR</td><td>13912-149&amp;nbsp;</td><td></td></tr><tr><td>RCT basic</td><td>IKA</td><td>3810001</td><td></td></tr><tr><td>Eppendorf Microcentrifuge 5424</td><td>VWR</td><td>80094-126</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>Millipore-Sigma</td><td>UFC901024</td><td></td></tr><tr><td>Allegra X-30 Series Benchtop Centrifuge</td><td>Beckman Coulter</td><td>B06314</td><td></td></tr><tr><td>UV-2600 Sptecrophotometer</td><td>Shimadzu</td><td>UV-2600 120V</td><td></td></tr><tr><td>Whatman Anotop 10 syringe filter</td><td>Sigma-Aldrich</td><td>WHA68091102</td><td></td></tr><tr><td>BD Disposable Syringes with Luer-Lok Tips</td><td>Fisher Scientific</td><td>14-829-45</td><td></td></tr><tr><td>Zetasizer Nano S</td><td>Malvern Panalytical</td><td>Zen 1600</td><td></td></tr><tr><td>1,1&amp;prime;-Dioctadecyl-3,3,3&amp;prime;,3&amp;prime;-tetramethylindocarbocyanine perchlorate</td><td>Sigma-Aldrich</td><td>42364-100MG</td><td></td></tr><tr><td>Dimethyl Sulfoxide, ACS</td><td>VWR</td><td>BDH1115-1LP</td><td></td></tr><tr><td>Sunshine Mix #1 LC1</td><td>Green Island Distributors, Inc</td><td>5212601.CFL080P</td><td></td></tr><tr><td>Adaptis 1000</td><td>Conviron</td><td>A1000</td><td></td></tr><tr><td>TES, &gt;99% (titration</td><td>Sigma-Aldrich</td><td>T1375-100G</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266-1KG</td><td></td></tr><tr><td>Air-Tite All-Plastic Norm-Ject Syringe</td><td>Fisher Scientific</td><td>14-817-25</td><td></td></tr><tr><td>Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers</td><td>Fisher Scientific</td><td>06-666A</td><td></td></tr><tr><td>Carolina Observation Gel</td><td>Carolina</td><td>132700</td><td></td></tr><tr><td>Corning microscope slides, frosted one side, one end</td><td>Sigma-Aldrich</td><td>CLS294875X25-72EA</td><td></td></tr><tr><td>Cork Borer Sets with Handles</td><td>Fisher Scientific</td><td>S50166A</td><td></td></tr><tr><td>Perfluorodecalin</td><td>Sigma-Aldrich</td><td>P9900-25G</td><td></td></tr><tr><td>Micro Cover Glasses, Square, No. 1</td><td>VWR</td><td>48366-045</td><td></td></tr><tr><td>Leica Laser Scanning Confocal Microscope TCS SP5</td><td>Leica Microsystems</td><td>TCS SP5</td><td></td></tr><tr><td>2&amp;prime;,7&amp;prime;-Dichlorofluorescin diacetate</td><td>Sigma-Aldrich</td><td>D6883-250MG</td><td></td></tr><tr><td>Dihydroethidium</td><td>Sigma-Aldrich</td><td>D7008-10MG</td><td></td></tr><tr><td>Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Eppendorf Uvette cuvettes</td><td>Sigma-Aldrich</td><td>Z605050-80EA</td><td></td></tr><tr><td>Chlorophyll meter&amp;nbsp;</td><td>Konica Minolta</td><td>SPAD-502</td><td></td></tr></tbody></table>

catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles

반응성 산소 종 (ROS) 축적 식물 abiotic 스트레스 반응의 특징 이다. 선생님 신호 수준 낮은 분자 및 높은 수준에서 분자를 손상 하 여 식물에는 이중 역할을 재생 합니다. 스트레스 식물에 선생님의 축적 대사, 효소, 지질, 및 식물 성장과 수확량의 감소를 일으키는 원인이 되는 DNA 손상 수 있습니다. 세 륨 산화물 나노 입자 (nanoceria)의 능력을 촉매로 선생님에서 vivo에서 청소는 독특한 도구 이해를 bioengineer 공장 abiotic 스트레스 관용을 제공 합니다. 여기, 선물이 합성 및 폴 리 (아크릴) 산 성 코팅된 nanoceria (PNC) 특성화, 식물 잎 lamina 침투를 통해 나노 인터페이스 및 그들의 분포와 선생님에서 vivo에서 confocal 사용 하 여 청소 하는 프로토콜 현미경 검사 법입니다. 식물에 선생님 축적을 조작 하기 위한 현재 분자 도구 모델 종으로 제한 하 고 힘 드는 변환 방법이 필요로. Vivo에서 선생님 청소에 대 한이 프로토콜 애기 thaliana같은 잎 구조와 넓은 잎 야생 타입 식물에 적용 될 가능성이 있다.

PNC 합성 및 특성 . PNC 종합 되었다 하 고 정화 방법을 프로토콜 섹션 2에서 설명 하는 다음 특징. 그림 1 A 세 륨 질산염, PAA, 세 륨 질산염 및 PAA, PNC의 혼합물의 솔루션의 채색을 보여 줍니다. PNC 합성 후 흰색에서 밝은 노란색 색상 변화를 볼 수 있다. 10 kDa 필터 정화, 후 피츠버그 대 일 분 광 광도와 특징 ...

Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

식물 반응 산소 종에서 Vivo에서 음이온 세 륨 산화물 나노 입자의 촉매 청소

여기, 선물이 vivo에서confocal 현미경 검사 법, 그리고 비보에 의해 식물 조직에서 이미징 nanoceria 청소 합성 및 선생님 (반응성 산소 종)에 대 한 세 륨 산화물 나노 입자 (nanoceria)의 특성에 대 한 프로토콜 nanoceria 선생님 confocal 현미경 검사 법에 의해 청소의 모니터링.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

catalytic-scavenging-plant-reactive-oxygen-species-vivo-anionic

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

8.9K 조회수.  University of California. 여기, 선물이 vivo에서confocal 현미경 검사 법, 그리고 비보에 의해 식물 조직에서 이미징 nanoceria 청소 합성 및 선생님 (반응성 산소 종)에 대 한 세 륨 산화물 나노 입자 (nanoceria)의 특성에 대 한 프로토콜 nanoceria 선생님 confocal 현미경 검사 법에 의해 청소의 모니터링.

비디오: Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

우라늄에서 구리 산화물 나노 입자에 의한 미량 원소의 제거<em&gt; 현장에서</em&gt; 복구 블리드 물과 세포 생존에 미치는 영향

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

연구

환경과학

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

메인 스트림 담배 연기에 자유 래디 칼을 감지하고 가스 단계 청소를위한 프로토콜

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

생체공학

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

의 CdSe @ CdS와 나노 막대에 이리듐 산화물 나노 입자의 광 화학적 산화 성장

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

화학

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

생화학

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical photocatalytic structures. However, the influence of such chemical bonding on the optical and surface properties of the photocatalyst and thus its photocatalytic activity/reaction selectivity behavior has not been systematically studied. In this investigation, TiO2 has been supported on the surface of SiO2 by means of two different methods: (i) by the in situ formation of TiO2 in the presence of sand quartz via a sol-gel method employing tetrabutyl orthotitanium (TBOT); and (ii) by binding the commercial TiO2 powder to quartz on a surface silica gel layer formed from the reaction of quartz with tetraethylorthosilicate (TEOS). For comparison, TiO2 nanoparticles were also deposited on the surfaces of a more reactive SiO2 prepared by a hydrolysis-controlled sol-gel technique as well as through a sol-gel route from TiO2 and SiO2 precursors. The combination of TiO2 and SiO2, through interfacial Ti-O-Si bonds, was confirmed by FTIR spectroscopy and the photocatalytic activities of the obtained composites were tested for photocatalytic degradation of NO according to the ISO standard method (ISO 22197&#8722;1). The electron microscope images of the obtained materials showed that variable photocatalyst coverage of the support surface can successfully be achieved but the photocatalytic activity towards NO removal was found to be affected by the preparation method and the nitrate selectivity is adversely affected by Ti-O-Si bonding.

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The focus of the present work is to establish means to generate and quantify levels of Ti-O-Si linkages and to correlate these with the photocatalytic properties of the supported TiO2.

TiO에서의 계면 화학 결합의 효과<sub&gt; 2</sub&gt; -SiO<sub&gt; 2</sub&gt; 광촉매 NOx 저감 성능 복합 재료

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

뉴클레아제 없는 산소 제거제 프로토카테의 발현 및 정제 3,4-디옥시게나아제

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

A Complete Method for Evaluating the Performance of Photocatalysts for the Degradation of Antibiotics in Environmental Remediation

Various antibiotics such as tetracycline, aureomycin, amoxicillin, and levofloxacin are found in large quantities in groundwater and soil systems, potentially leading to the development of resistant and multi-drug resistant bacteria, posing a threat to humans, animals, and environmental systems. Photocatalytic technology has attracted keen interest due to its rapid and stable treatment and direct use of solar energy. However, most studies evaluating the performance of semiconductor catalysts for the photocatalytic degradation of organic pollutants in water are currently incomplete. In this paper, a complete experimental protocol is designed to comprehensively evaluate the photocatalytic performance of semiconductor catalysts. Herein, rhombic dodecahedral silver phosphate was prepared by a simple solvent phase synthesis method at room temperature and atmospheric pressure. BrSubphthalocyanine/Ag3PO4 heterojunction materials were prepared by the solvothermal method. The catalytic performance of as-prepared materials for the degradation of tetracycline was evaluated by studying different influencing factors such as catalyst dosage, temperature, pH, and anions at atmospheric pressure using a 300 W xenon lamp as a simulated solar light source and a light intensity of 350 mW/cm2. Compared with the first cycle, the constructed BrSubphthalocyanine/Ag3PO4 maintained 82.0% of the original photocatalytic activity after five photocatalytic cycles, while the pristine Ag3PO4 maintained only 28.6%. The stability of silver phosphate samples was further tested by a five-cycle experiment. This paper provides a complete process for evaluating the catalytic performance of semiconductor catalysts in the laboratory for the development of semiconductor catalysts with potential for practical applications.

Presented here is a protocol to explore a universal set of experimental procedures for comprehensive laboratory evaluation of photocatalysts in the field of environmental purification, using the example of photocatalytic removal of antibiotic organic pollutant molecules from water by phthalocyanine sensitized silver phosphate composites.

환경 개선에서 항생제 분해를 위한 광촉매의 성능을 평가하기 위한 완전한 방법

Various antibiotics such as tetracycline, aureomycin, amoxicillin, and levofloxacin are found in large quantities in groundwater and soil systems, ...

A Continuous-flow Photocatalytic Reactor for the Precisely Controlled Deposition of Metallic Nanoparticles

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic nanoparticles is developed. Guidelines for the replication of the reactor and its operation are provided in detail. Three different composite systems (Pt/graphene, Pt/TiO2, and Au/TiO2) with monodisperse and uniformly distributed particles are produced by this reactor, and the photodeposition mechanism, as well as the synthesis optimization strategy, are discussed. The synthesis methods and their technical aspects are described comprehensively. The role of the ultraviolet (UV) dose (in each excitation pulse) on the photodeposition process is investigated and the optimum values for each composite system are provided.

For a continuous and scalable synthesis of noble-metal-based nanocomposites, a novel photocatalytic reactor is developed and its structure, operation principles, and product quality optimization strategies are described.

금속 나노 입자의 정확 하 게 제어 증 착에 대 한 연속 흐름 촉매 반응 기

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic ...

Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. Traditionally, they were synthesized through thermal cycloaddition reactions, whereas today, photocatalysis is preferred due to the mild and efficient conditions. With this focus, a new photocatalytic method for the synthesis of nitrogen-containing heterocycles is highly desired. Here, we report a protocol for the biosynthesis of cercosporin, which could function as a metal-free photocatalyst. We then illustrate cercosporin-photocatalyzed protocols for the synthesis of nitrogen-containing heterocycles 1,2,3-thiadiazoles through annulation of azoalkenes with KSCN, and synthesis of 1,4,5,6-tetrahydropyridazines [4+2] through cyclodimerization of azoalkenes under mild conditions, respectively. As a result, there is a new bridge between the microbial fermentation method and organic synthesis in a mild, cost-effective, environmentally friendly and sustainable manner.

New routes for the synthesis of nitrogen-containing heterocycles utilizing cercosporin as a metal-free photocatalyst were developed.

세르코스포린-포토카탈루즈 [4+1]- 및 [4+2]-온화한 조건하에서 아졸케네스의 탄화

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

질소 산화물과 세포로부터 전자 상자 성체의 프로그램 공명 분광학에 의한 초과 산화물 음이온 급진적가 탐지 스핀 트랩을 사용하여

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...