This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1. 광학 보정 : 측정 광 출력 <ol><li> 광 전달 장치를 준비하기 위해, 광대역 광원을 연결 (예를 들어, 제논 램프 또는 텅스텐)에 적합한 전원 공급 장치에 관한 것이다. 광의 시준 된 빔을 생성하는 광원 앞의 콜리 메이팅 렌즈를 배치. 광속에서 대부분의 열을 제거하기 위해 액체 열 필터를 통해 광을 통과. 애플리케이션에 따라, 빛의보다 유연한 전달 (예., 인큐베이터) 제공 액체 광 가이드의 입사 개구에 평행 빔에 초점. </li><li> 액체 광 가이드의 타단에, 시준 렌즈를 제하고 간섭 및 감광 필터의 위치 홀더. 이 배열은 원하는 파장 대역과 강도의 빛의 균일 조명 자리를 생산하고 있는지 확인합니다. 참고 : 라이트 가이드와 시편 평면의 끝 사이의 거리에 따라 달라질 필요가 있습니다조명되어야하지만 지역 도광 증가 단부로부터의 거리로, 광 강도가 감소한다는 것을 기억. 본 연구에서는,이 거리가 14cm이고 균일 96- 웰 플레이트에 3 × 3 웰 영역을 조명 빔 단면을 생성한다. </li><li> 램프와 관련 광학 부품으로부터 미광을 확인하는 것은 시편에 도달 할 수 없습니다. </li><li> 액체 열 필터 물 쿨러의 전원을 켜고 필터 재킷을 통해 물 교환이 확인합니다. 램프 전원을 켜고 안정 될 때까지 광대역 광원 적어도 5 분 동안 기다립니다. 주 : 물 냉각기의 사용은 장치의 과열을 방지 할 필요가있다. </li><li> 조명의 원하는 파장 대역을 생성하는 (보통 반 최대 (FWHM) 대역폭에서 자신의 중심 파장, 피크 투과율 및 전체 폭에 의해 기술) 협 대역 간섭 필터를 선택합니다. 참고 : 본 연구에 사용 된 협 대역 간섭 필터 (4)이었다42 나노 미터, 550 나노 미터, 671 nm에서 810 nm의. </li><li> 시편은 치료 중에 위치하도록 평면에서 램프에 의해 생성 된 광을 측정한다. 제조업체의 지시에 따라 적부 소프트웨어를 사용하여, 적절한 접속 분광 방사 조도 교정 프로브 (코사인 콜렉터)을 사용하여 광을 측정한다. </li><li> 원하는 출력이 얻어 질 때까지의 광의 강도를 조정하는 감광 필터를 사용한다. 참고 : 본 연구에서, 각각의 간섭 필터에 의해 통과 된 광의 강도는 네 개의 처리 wavebands 각각에서 동일한 계량 적 출력을 제공하도록 조정된다. 이 램프 출력이 변경 될 수 있습니다로, 정기적으로 교정을 확인하는 것이 중요합니다. 주 : 장치의 셋업이 이어, 세포를 조명 될 준비가 하나가 현재 연구에 사용 된 광 전달 장치의 그림 표현이다.. </li></ol><img alt="그림 1"SRC = &quot;/ 파일 / ftp_upload / 52221 / 52221fig1.jpg&quot;/&gt; 빛 전달 장치. 일러스트 그림 1. 이미지는 빛 전원 주택, 시준 렌즈, 물 필터, 입구 조리개, 액체 라이트 가이드, 초 콜리 메이팅 렌즈, 필터 홀더, 처리 프레임과 매트 블랙 카드 크세논 램프입니다. 협 대역의 파장과 강도 필터가 표시되지 않습니다. 2. 셀 준비 <ol><li> 기재된 R / NIR-LT 배송 방법은 어떠한 세포 유형 또는 시험 관내 모델 시스템에 적용될 수있다; 세포 배양의 다음과 같은 설명은 배양 갈색 세포종 (PC12), 뮐러 (RMC1)와 일차 혼합 망막 세포에 잘 확립 된 기술을 사용하여, 일반적으로이다. </li><li> 빛 치료 및 산화 스트레스 분석을위한 세포 파종하기 전에, 문화는 T75의 flas에서 적절한 보조제 (예., FBS, 항생제)를 포함하는 각각의 성장 배지에서 세포를 불멸화70-80% 합류 할 때까지 KS. </li><li> 예를 테스트하기 위해 세포 유형에 적합한 방법을 사용하여 T75 플라스크로부터 불사 화 세포를 분리., 트립신. 필요한 경우, 96 웰 분석 플레이트에서 세포의 파종 진행하기 전에, 코트 분석 플레이트 (PC12 세포에 대한 예.) 1 시간에 대한 10μg / ml의 폴리 -L- 라이신과 우물 또는 10μg / ml의 폴리 -L- 라이신 10μg와 O / N 배양 한 다음 1 시간 / ㎖ 라미닌 (예., 혼합 망막 세포). </li><li> 원심 분리 세포 (예., PC12 세포에 대한 405 XG 또는 RMC1 전지용 218 XG에 10 분에서 3 분)와 같은 적절한 수집 할 적절한 세포 배양 배지 8 ㎖에 상청에 resuspend 제거. </li><li> 혼합 망막 세포가 사용되는 경우, 확립 된 절차에 따라 19 소화 효소 (파파인)에 의해 P0-5 신생아 래트 새끼로부터 제조 </li><li> 혈구 계를 사용하여, 0.4 % (W / V) 트립 판 블루 염색 배제 생존 세포 수를 계산. </li><li> 셀 밀도 SUC를 조정세포 배양에서 24 또는 48 시간 후 약 70~80% 합류 될 것입니다 시간. (PC12 세포에 대한 시딩 밀도 RMC1 세포를 들어, 2.5 × 10 5 생세포 / ㎖이고, 혼합 망막 세포 것이 8 × 10 5 생세포 / ㎖이며, 4.0 × 10 5 생세포 / ㎖ 임). 셀을 추가 한 후 중 적절한 성장 배지 100 ㎕에, (H 2 O 2 또는 DCFH-DA 분석 각각 사용) 명확한 또는 검은 색 96 웰 플레이트, 플레이트는 37 ° C, 5 평평한 표면에 앉아 할 수 있도록 % CO 2 세포 부착을 허용하는 최소 24 시간 동안 (또는 어떤 조건은 특정 세포 유형에 적합한). </li><li> 해당 96 웰 트레이 성장 미디어 문화의 세포들은 70 ~ 80 %의 합류 (PC12 및 RMC1 세포에 대한 24 시간, 혼합 망막 세포 48)까지. </li></ol> 3. 세포에 글루타민산 스트레스 추가 <ol><li> 적절한 성장한다 전체에 0-10mM의 L 글루탐산 모노 나트륨 염 수화물 스트레스 농도 준비일 문화 미디어. </li><li> 세포에서 미디어를 제거하고 부드럽게 PBS (PC12) 또는 HBSS (RMC1과 혼합 된 망막 세포)와 세포를 3 회 세척한다. 세척 후 / 웰에 100㎕의 총 부피로 글루타메이트 - 함유 배지를 추가한다. </li></ol> 라이트 치료 4. 첫 번째 투여 량 <ol><li> 즉시 글루타메이트 또는 선택의 스트레스 요인의 추가에 따라, 제조 된 광 전달 장치 아래에 배치하여 원하는 파장과 강도 빛 치료에 세포를 노출합니다. 파장에 따라 중립적 인 밀도 필터의 설치 조합을 사용하여 광선의 계량 적 출력 관리중인 변경해야합니다. 참고 : 현재의 실험에서, 3 분 동안 빛 치료에 세포를 노출 37 O C 열 패드의 96 웰 플레이트를 놓아 온도를 유지한다. 요구되는 처리 시간은 변할 수있다. <ol><li> 96 아 인접 우물에 반영하는 빛을 방지하기 위해 세포 아래에 매트 블랙 카드를 놓습니다L 트레이는 다른 치료 매개 변수에 대한 지정. </li></ol></li><li> 처리는 시간의 긴 길이에 대해 요구되는 경우, 최적 또는 조건을 세포 배양 배지의 pH를 유지 또는 이상적으로, 5 % CO 2로 조절 CO 2 농도 인큐베이터 내에서 장치 및 처리 판을 배치하고 25mM HEPES 완충 추가 특정 세포 유형. </li><li> 광처리 종료 후, 평평한 표면에 세포를 넣고 37 ℃, 5 % CO 2에서 24 시간 배양한다 대 (또는 어떤 상황 세포 유형에 대해 최적). 주의 : 전술 한 바와 같이 필요에 따라 R / NIR-LT의 추가의 투여 량은, 투여 할 수있다. </li></ol> ROS의 빛 치료와 탐지 5. 최종 복용량 <ol><li> 광처리의 최종 라운드를 수행하기 전에 시약 ROS 검출에 사용될 준비. 2 O 2 검출 시약 작업을 50mM의 구연산 완충액 (pH6.0), 트리톤 X100 및 H 준비용액 (1 : 2 : 10 mM이 H 2 O 2 검출 시약 원액의 비율 97 10 U / ㎖의 양 고추 냉이 퍼 옥시 다제, 50 mM의 시트르산 나트륨 (PH 6.0)). 적절한 미디어에서 100μM의 최종 농도 DCFH-DA를 준비합니다. 주 : PC12 세포에 DCFH-DA를위한 시약 RPMI 매체이며, RMC1 전지용 DCFH-DA 시약 DMEM 배지에서이다. 시판 검출 시약 특정 ROS에 차동 감도를 가지고 시약 개별 애플리케이션에 요구되는 정보를 제공하기 위해주의 깊게 선택되어야합니다. </li><li> 4.1-4.1.2 단계에 기재된 바와 같이, 세포에 광처리를 관리. 확인 세포는 빛의 원하는 플루 언스 / 파장으로 처리되고있다. </li><li> 즉시 글루타메이트 - 함유 배지를 제거하고 적절한 완충 용액으로 두 번 세척 광처리는 다음의 (PC12 세포 위해 PBS를 사용하고, 셀 RMC1 위해 HBSS가 사용된다). 다음과 같이 세포의 ROS 분석을 수행합니다 : <ol><li> H 2 O 2 </suB&gt; 분석은 : 각각의 웰에 50 mM의 구연산 완충액 (pH 6.0) 및 5 μL의 트리톤 X100 45 μL를 추가한다. 부드럽게 30 초 동안 진탕 기에서 세포를 흔들어 15 분 동안 CO 2를 37 ° C에서 품어 5 %. H의 50 μL 2 O 2 탐지 작업 솔루션을 추가하고 실온에서 30 분 동안 품어. 을 530nm의 여기 파장 및 ~ 480㎚의 발광 파장을 갖는 플레이트 판독기를 사용하여 형광을 측정한다. </li><li> DCF 분석은 : 각각의 웰에 DCFH-DA의 100 μM 용액 100 μl를 추가하고 37 ° C에서 30 분, 5 % CO 2 배양한다. 100 μM DCFH-DA가 포함 된 용지를 제거하고 위에서 설명한대로 두 번 적절한 완충 용액으로 세척한다. 각각의 웰에 완충 용액 10 μL 트리톤 X100의 90 μl를 추가하고 부드럽게 37 ° C에서 30 초 15 분 전에 배양을위한 궤도 진탕 기에서 흔들어. ~ 480㎚의 여기 파장과 발광 wavel와 플레이트 판독기를 이용하여 유도 된 DCF 형광 측정을 530nm의 ength. </li><li> 익스프레스 ROS는 단백질 MG를 계산하는 표준 곡선을 참조하여, 제조업체의 지시에 따라 단백질 농도를 비색 정량 키트를 이용하여 웰에 남아있는 세포의 단백질 농도에 대하여 값. </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+near-infrared+laser+therapy+applied+to+promote+clinical+recovery+in+acute+and+chronic+neurodegenerative+diseases.">Transcranial near-infrared laser therapy applied to promote clinical recovery in acute and chronic neurodegenerative diseases.</a> Expert review of medical devices. 9, 71-83 (2012).</li><li>Chung, 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=The+nuts+and+bolts+of+low-level+laser+(light)+therapy.">The nuts and bolts of low-level laser (light) therapy.</a> Annals of biomedical engineering. 40, 516-533 (2012).</li><li>Wu, Q., 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=Low-Level+Laser+Therapy+for+Closed-Head+Traumatic+Brain+Injury+in+Mice:+Effect+of+Different+Wavelengths.">Low-Level Laser Therapy for Closed-Head Traumatic Brain Injury in Mice: Effect of Different Wavelengths.</a> Lasers in surgery and medicine. 44, 218-226 (2012).</li><li>Hashmi, J. T., 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=Role+of+low-level+laser+therapy+in+neurorehabilitation.">Role of low-level laser therapy in neurorehabilitation.</a> PM&amp;R. 2, S292-S305 (2010).</li><li>Fitzgerald, M., 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=Metallothionein-IIA+promotes+neurite+growth+via+the+megalin+receptor.">Metallothionein-IIA promotes neurite growth via the megalin receptor.</a> Experimental Brain Research. 183, 171-180 (2007).</li><li>Fitzgerald, M., 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=Near+infrared+light+reduces+oxidative+stress+and+preserves+function+in+CNS+tissue+vulnerable+to+secondary+degeneration+following+partial+transection+of+the+optic+nerve.">Near infrared light reduces oxidative stress and preserves function in CNS tissue vulnerable to secondary degeneration following partial transection of the optic nerve.</a> Journal of neurotrauma. 27, 2107-2119 (2010).</li><li>Ando, T., 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=Low-level+laser+therapy+for+spinal+cord+injury+in+rats:+effects+of+polarization.">Low-level laser therapy for spinal cord injury in rats: effects of polarization.</a> Journal of biomedical. 18, 098002 (2013).</li><li>Lanzafame, R. J., 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=Reciprocity+of+exposure+time+and+irradiance+on+energy+density+during+photoradiation+on+wound+healing+in+a+murine+pressure+ulcer+model.">Reciprocity of exposure time and irradiance on energy density during photoradiation on wound healing in a murine pressure ulcer model.</a> Lasers in surgery and medicine. 39, 534-542 (2007).</li><li>Castano, A. P., 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=Low-level+laser+therapy+for+zymosan+induced+arthritis+in+rats:+Importance+of+illumination+time.">Low-level laser therapy for zymosan induced arthritis in rats: Importance of illumination time.</a> Lasers in surgery and medicine. 39, 543-550 (2007).</li></ol>

The authors declare that they have no competing financial interests.

우리는 성공적이 R / NIR-LT 시험관 최적화 연구기구를 제공하기 위해 정밀한 보정 광 전송 시스템을 채택했다. R의 파장 및 강도 파라미터 / NIR-LT는이 시스템을 이용하여 정확하고 효율적으로 조작 될 수있다. ROS 테스트 세포 유형에서 제공되는 파장과 투여 량 감소되지 않았다하더라도 우리는 세포 죽음으로 이어질하지 않은 세포의 빛 치료를 설립했다. 670nm (20.11W / m 2)에서 현재 시?...

반응성 산소 종 (ROS)의 신호 전달 경로와 하나의 신경 세포를 포함한 정상적인 대사 반응의 범위에 대해 요구된다. 내인성 항산화 메커니즘은 ROS의 생성을 제어 할 수없는 때, 세포는 산화 스트레스에 굴복 2,3-있다. CNS에 손상 후, ROS 및 산화 스트레스의 존재가 증가 연관된 손상 4,5의 진행에 중요한 역할을하는 것으로 생각된다. 평가 된 산화 스트레스를 감쇠하기위한 전략의 광대 한 수에도 불구하고, 신경 외상 (6) 다음의 임상 사용 ROS 생산과 관련된 산화 스트레스를 감쇠에 대한 완전히 효과, 임상 적으로 관련된 항산화 전략은 현재 존재하지 않는다. 따라서 산화 스트레스의 감쇠는 치료 적 개입 7에 대한 중요한 목표 남아있다. 개선 사항은 다음과보내고 R / NIR-LT는 부상 아마도 산화 스트레스를 줄임으로써 심근 경색 크기, 당뇨병 및 간 동안 신장 합병증, 망막 변성, CNS 손상 및 뇌졸중 (8)의 감소를 포함하여 다양한 질환에서보고되었다. CNS 부상에 특히 관련하여, 670nm의 빛의 효능 임상 연구는 망막 변성 9-11, 척수 손상 (12), 신경 세포의 죽음 (13)의 모델에 좋은 효과를 보여 주었다. 임상 시험은 그러나이 실험의 결과는 15 매개 변수 효과적인 치료를 사용하는 것이 아마도 장애로 인해 유망 나타나지 않습니다, 황반 변성 관련 마른 나이 수행과 뇌졸중 (14)에 대해 현재 진행되고있다. 따라서, R / NIR-LT 널리 비 침습적, 관리하기 쉽고 상대적으로 저렴한 치료에도 불구하고, 신경 외상 정상 임상 실습의 일환으로 채택되지 않았습니다. 임상 번역 장벽은 CL의 부족을 포함초기 표준화 효과적인 치료 프로토콜 16, 17의 행동과 부재의 메커니즘을 이해했다. 빛 치료에 대한 현재 문헌 조사 소스에 대해 (LED 또는 레이저), 파장 (예를 들어, 630, 670, 780, 810, 830, 880, 904nm), 조사의 총 용량 (주울 / 치료 매개 변수의 변화의 과다를 보여 단위 영역), 지속 시간 (노광 시간), 타이밍 (예정 모독 또는 포스트), 치료의 빈도 및 전달 모드 () 펄스 또는 연속 8. 연구와 치료 매개 변수의 변동성 비교 어렵고 효능 (16)에 대한 회의론에 기여하고있다. 따라서, R / NIR-LT의 최적화는 명확 여러 변수를 비교하는 데 필요한 높은 처리량 스크리닝 기법을 제공 할 수있는 세포 배양 시스템이 필요하다. 그러나 워싱턴를 통해 충분한 유연성과 제어를 제공 할 수 몇 상업적으로 이용 가능한 조명 시스템이 있습니다velength 강도는 최적화 실험을 수행 할 수 있습니다. 시판 LED 장치는 일반적으로 강도뿐만 아니라 다를 수 있습니다 다른 제조 업체, 여러 LED 소자를 사용하는 연구자의 결과로 여러 파장이나 강도를 제공 할 수뿐만 아니라 방출되는 빛의 파장의 스펙트럼하지 않습니다. 우리는 따라서 R / NIR-LT의 파라미터의 근접, 정확한 제어를 허용하며, 파장 및 빛의 플루 언스의 범위를 생성하는 협 대역 간섭 필터를 통해 여과 광대역 크세논 광원을 사용함으로써이 문제를 해결했다. 그것은 치료의 치료 학적 투여 량은 R / NIR-LT의 경우 사이토 크롬 C 산화 효소 (COX) (18)로 가정하고, photoacceptor (발색단)와 상호 작용하는 광자의 수에 의해 정의된다는 점에주의하는 것이 중요하다. 전달 광자 에너지는 파장에 따라 달라집니다; 서로 다른 파장의 에너지의 동일한 용량을 의미하는 것은 COM 될 것입니다광자의 상이한 수의 입상. 따라서, 소자로부터 방출 된 광이 테스트에 선택된 파장의 광자를 각각 동일한 수의 방출하도록 조정 하였다. 생체 외에서 세포의 파장과 강도의 범위에서 R / NIR-LT를 전달하는데 사용될 수있는 시스템을 개발하고 실시 세포에서 ROS 생성에 전달되는 R / NIR-LT의 효과를 측정 할 수있는 능력을 입증 글루타민산 염 스트레스.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

레이저 또는 발광 다이오드 (LED)에 의해 전달 레드 / 근적외선 요법 (R / NIR-LT)은, 아마도 산화 스트레스를 감소시킴으로써 생체 내에서 중추 신경계 손상, 범위의 기능적 형태 학적 결과를 개선시킨다. 그러나, 산화 스트레스에 R / NIR-LT의 효과는 파장 또는 조사의 강도에 따라 달라질 것으로 나타났다. 치료 매개 변수를 비교하는 연구로 인해 체외 최적화 연구에 넣어 통해 높은에 적합한 여러 파장이나 강도를 제공 상업적으로 사용 가능한 장치의 부재로, 부족하다. 이 프로토콜은 주어진 손상 모델에 필요한 치료 용량을 최적화하는 파장과 강도의 범위에서 빛의 전달을위한 기술을 설명한다. 우리는 파장 및 강도 파라미터를 쉽게 변경 될 수있는 빛을 전달하는 방법은, 활성 산소 종을 감소시키기위한 R / NIR-LT의 최적 투여 량의 결정을 용이하게 할 수 있다는 가설을 세웠다시험 관내 (ROS). 비 간섭 제논 라이트는 다양한 파장 (440, 550, 670의 중심 파장 및 810) 및 플루 언스 제공하는 협 대역 간섭 필터를 통해 여과 (8.5 × 10 -3 3.8을 × 10 -1 J / cm 2) 빛의 배양 된 세포에. 장치에서 빛 출력은 각각의 파장에서 빛의 치료 관련, 동일한 계량 적 투여 량을 방출하도록 조정했다. 글루타메이트는 DCFH-DA 및 H 2 O 2에 민감한 형광 염료를 사용하여, 광 처리 된 세포 스트레스에 반응 종을 검출 하였다. 우리는 성공적으로 CNS 손상의 모델로 글루타민산 염에 노출 배양 된 세포에 관련 파장 및 강도 생리 및 치료의 범위에서 빛을 전달했다. 배양 된 세포에 의해 생성 된 ROS에 영향을주지 않았다 현재 연구에 사용 된 R / NIR-LT의 플루 언스 반면, 빛의 전달 방법은 다른 SYST에 적용 할 수있다EMS 더 중요한 생리 학적 또는 미토콘드리아 Organotypic 슬라이스 배양 모델을 포함한 절연 및 산화 대사의 결과 측정의 범위에 영향을 평가하기 위해 사용될 수있다.

670nm의 파장에서 전달되는 빛의 출력은 이전에 생체 내 (0.3 J / cm 2) (20)에 유익한 것으로 도시 670nm 광의 도즈 포괄 플루 언스의 범위로 셀을 조사하기 위해 감광 필터를 사용하여 보정 하였다. 증가 된 광원의 앞에 중성 밀도 필터의 개수, 강도 (W / m 2) 이하 광 표적 영역에 전달할 수 있도록, 감소 하였다. 표 1은 파장 필터 장착 광원으로부터 발생 670nm 광의 교정 데이터...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

파장과 산화 스트레스에 레드 / 근적외선 치료의 강렬의 범위의 효과의 평가를위한 방법<em&gt; 체외</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

8.6K 조회수.  The University of Western Australia. Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

비디오: Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

라이트 농도의 변화에​​ Zebrafish의 응답을 측정하기 위하여 행동 어세이

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

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Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

글리코겐의 생화학 적정<em&gt; 체외</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

제브라 피쉬 배아에서 산화 스트레스의 분석

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

의 산화 스트레스 저항을 측정<em&gt; 예쁜 꼬마 선충</em&gt; 96 웰 마이크로 타이 터 플레이트에

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

내피 세포 접착 정량<em&gt; 체외</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

갭 접합부 세포 간 통신 : 천연 제품의 독성 물질과 독소의 반작용을 평가하기위한 기능성 바이오 마커, 보건 혜택

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP 현미경을 이용한 miRNA의 gap junction 의존적 전달 분석

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

마우스의 장내 미생물에 대한 박테리오신 효과 평가 방법

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

해로운 효과의 낮은 압력 플라즈마 살 균 새 균의 subtilis 포자를 사용 하 여 라이브 셀 현미경의 생존에 조사

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...