1. 악기 설정 <ol><li> spectrofluorometer 켜고 1 시간 동안 워밍업 수 있습니다. </li><li> Excel이 그림 1에 나타난 표준 및 샘플 데이터 파일을 만들고 분리 쉼표에 저장 사용하면 &quot;CSV&quot;형식을 값. <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="그림 1" /> 그림 1. 표준 및 샘플 데이터입니다. </li><li> 다음과 같이 fluorometer를 설정합니다 : <ol><li> 을 클릭 <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="아이콘 1" /> 버튼이 그림 2에 표시된 분석 방법 창이 열립니다. <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="그림 2" /> 그림 2. 분석 방법 윈도우, 일반 탭을 클릭합니다. 다음과 같이 선택합니다 : <ul><li> &quot;측정&quot;필드에서 풀다운 메뉴에서 측광을 선택합니다. </li><li> &quot;연산자&quot;입력란에 해당 운영자의 이름을 입력합니다. </li><li> &quot;악기&quot;입력란에 정보를 입력할 필요가 없습니다. 이것은 소프트웨어에 의해 자동으로 선택됩니다. </li><li> microplate 리더를 사용할 때 &quot;샘플링&quot;필드는 비활성화되어 있습니다. </li><li> 당신이 &quot;의견&quot;입력란에 리포트에 포함시킬 수있는 의견을 입력하십시오. </li><li> &quot;액세서리&quot;입력란에이 시험에 사용되는 부속품에 관한 정보를 입력하십시오. </li><li> 창의 오른쪽에있는 버튼을합니다. </li><li> &quot;로드&quot;버튼을 이전에 저장한 분석 방법을 로딩 있습니다. </li><li> &#39;저장&#39;버튼은 동일한 이름을 사용하여 테스트 매개 변수 또는 이전에 저장한 및로드 분석 방법의 집합을 업데이 트하실 수 있습니다. </li><li> 버튼을 원하는 이름으로 테스트 매개 변수 또는 분석 방법의 새로운 집합을 저장 가능 &quot;다른 이름으로 저장&quot;. </li></ul> 이제 &quot;Quantitation&quot;탭을 클릭, 그림 3을 참조하십시오 다음과 같은 선택합니다. <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="그림 3" /> 그림 3. 분석 방법 윈도우, Quantitation 탭. <ul><li> &quot;Quantitation 형식&quot;입력란에 파장을 선택합니다. 이것은 형광 읽기가 선택한 파장을 사용하여 이루어집니다 나타냅니다. </li><li> &quot;보정 형식&quot;입력란에 1 일 순서를 선택합니다. </li><li> &quot;파장 번호 &#39;입력란에 1을 선택합니다. </li><li> &quot;농도 단위&quot;필드에서 NG / ML을 입력하십시오. </li><li> &quot;수동 보정&quot;와 &quot;제로를 통해 강제로 곡선&quot;에 대한 확인란을 선택되지 않은 둡니다. </li><li> 입력란에 &quot;소수점 뒤 자리&quot;에서는, 2를 선택합니다. </li><li> &quot;로우어 농도 제한&quot;필드에 0을 입력합니다. </li><li> &quot;어퍼 농도 제한 필드에 10000을 입력합니다. </li></ul> 이제 &#39;악기&#39;탭을 클릭하고 다음과 같이 선택합니다. <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="그림 4" /> 그림 4. 분석 방법 창, 인스 트루먼트 탭 <ul><li> &quot;데이터 모드 :&quot;입력란에 형광을 선택합니다. </li><li> &quot;초핑 속도&quot;필드는이 시간에, 그것이 인광 측정에 대해서만 사용되는 운영 중지 상태입니다. </li><li> &quot;파장 모드&quot;필드에서 WL 고정 모두를 선택합니다. </li><li> &quot;EX / WL1 &#39;입력란에 480nm 입력하십시오. </li><li> &quot;EM / WL1&quot;필드에서 520nm를 입력하십시오. </li><li> 다른 모든 EX 아래의 필드와 EM은 현재 운영 중지 상태입니다. </li><li> &quot;EX 슬릿 &#39;입력란에 풀다운 메뉴에서 5.0nm 선택합니다. </li><li> &quot;EM 슬릿 &#39;입력란에 풀다운 메뉴에서 5.0nm 선택합니다. </li><li> &quot;PMT 전압 1 - 1000V &#39;필드 앞에있는 확인란을 선택되지 않은 둡니다. </li><li> &quot;PMT 전압&quot;필드에서 풀다운 메뉴에서 700V를 선택합니다. </li><li> &quot;자동 통계 CALC. 번호&quot;입력란에 2를 선택합니다. </li><li> &quot;복제&quot;입력란에 1을 선택합니다. </li><li> &quot;통합 시간&quot;필드에서 0.1s를 선택합니다. &lt;은/ 리&gt; <li> &quot;지연&quot;입력란에 입력하거나 개로 선택하십시오. </li></ul> &#39;모니터&#39;탭을 클릭하십시오. <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="그림 5" /> 그림 5. 분석 방법 창, 모니터 탭을 클릭하십시오. <ul><li> &quot;Y 축&quot;에서 &quot;최대 :&quot;입력란에 10000을 선택합니다 </li><li> 에서 &quot;Y 축&quot;, &quot;최소 :&quot;필드에 0을 선택합니다. </li><li> &quot;데이터 수집 후 처리 오픈 데이터&quot;의 왼쪽에있는 확인란을 선택합니다. </li><li> 이 수치가 완료된 후 자동으로 인쇄하기 위해 리포트를 원하지 않으시면 &#39;데이터 수집 후 인쇄 보고서 &quot;의 왼쪽에있는 확인란을 선택되지 않은 둡니다. </li></ul> &#39;보고서&#39;탭을 클릭, 그림 6을 참조하십시오. <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="그림 6" /> 그림 6. 분석 방법 창, 보고서 탭. <ul><li> &quot;출력&quot;필드에서 풀다운 메뉴에서 인쇄 보고서를 선택합니다. 당신은 또한 사용자 정의 만든 리포트를 생성할 수 있습니다 프로그램 보고서 생성기에 선택 추가를 사용하는 경우 Excel로 내보낸 데이터 또는 &quot;사용 인쇄 생성기 시트&quot;를 원한다면 &quot;Microsoft Excel을 사용&quot;을 선택 수 있습니다. </li><li> 이 보고서에 포함 시키고자하는 항목에 대한 상자를 클릭합니다. </li><li> &quot;분석 방법&quot;윈도우의 여러 탭에서 선택한 모든 매개 변수를 저장하려면, &quot;&quot;일반 &quot;탭을 다시 클릭하고 파일 이름으로하고 나중에 사용하기 위해 그것들을 찾을 수있는 위치에 그들을 저장하고,를 클릭 확인 &quot;이 창을 닫습니다 버튼을 누르십시오. </li><li> 이것은 악기 테스트 매개 변수를 설정 완료됩니다. </li><li> 이제 우리는 microplate 리더를 설치합니다. </li><li> 을 클릭 <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="아이콘 2" /> 위에 MPR 설치 윈도우가 나타납니다 버튼. </li></ul> 그림 7에 표시된 MPR 설치 윈도우 / 플레이트 탭에서 표준 및 샘플을위한 우물을 선택합니다. <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="그림 7" /> 그림 7. MPR 설정 창, 플레이트 탭을 클릭하십시오. <ul><li> A1 위치를 클릭하고 E1 마우스를 드래그 다음을 클릭합니다 <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="아이콘 3" /> 버튼을 클릭합니다. 이것은 표준에 사용되는 위치를 선택합니다. 이러한 순위는 녹색으로 강조 표시됩니다. </li><li> 이제 우리는 샘플 H3에 위치의 F1을 선택합니다. 를 누른 다음를 클릭하고 위치 F1부터 마우스를 드래그 H3 위치를하고 <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="아이콘 4" /> 버튼이 노란색으로 그 위치를 강조 표시됩니다. </li><li> 다음 샘플 측정에 그 위치를 선택하고, E3에 A2 위치에 대해 동일한 작업을 반복합니다. </li><li> 이제 변수 (CSV) 파일을 구분된 파일 쉼표로 표준 및 샘플 정보 사전에 준비를로드하자. </li></ul> 그림 8 표시된 &quot;MPR 설정 창&quot;의 &quot;자 정보&quot;탭을 클릭합니다. <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="그림 8" /> 그림 8. MPR 설정 창, 웰 정보 탭을 클릭하십시오. <ul><li> 다음을 클릭합니다 <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="아이콘 5" /> 버튼을 클릭합니다. Windows 탐색기 창을은 &quot;글쎄 information.csv&quot;파일을 찾는 데 그것을 로딩 있도록 그림 9에 표시하는 열립니다. <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="그림 9" /> 그림 9. 물론 샘플 정보를로드 중입니다. &quot;자 정보&quot;파일을로드 후 &quot;그럼 정보는&quot;그림 10에서 그림과 같이 표시됩니다 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="그림 10" /> 그림 10. 샘플 잘 정보를 로드된. </li></ul></li></ol></li></ol> 2. Picogreen 분석 샘플 준비 <ol><li> 20 X TE 버퍼 1 ML 추가하여 1X TE 버퍼 (10 MM 트리스 - HCL, 1 MM EDTA (에틸렌 다이아 민 테트라 초산)) 준비19 ML DI H 2 O. </li><li> 9.95 ML 1 X TE 버퍼 50 μL 200 X PicoGreen 시약을 추가하여 PicoGreen 시약 솔루션을 준비합니다. </li><li> 주식 dsDNA (100 μg / ML)의 시리얼 dilutions함으로써 표준 (람다 dsDNA)를 준비합니다. 먼저 주식 dsDNA (100 μg / ML) 25 μL 1 X TE 버퍼 25 μL를 추가하여 50 μg / ML dsDNA의 솔루션을 준비합니다. 그런 다음 아래의 표에 표시된 dsDNA의 볼륨과 버퍼를 추가하여 표준 솔루션을 준비 : <table border="1"><tbody><tr><td> dsDNA의 농도 </td><td> dsDNA의 볼륨 </td><td> 버퍼의 볼륨 </td></tr><tr><td> 1 μg / ML </td><td> 50 μg / ML dsDNA 20 μL </td><td> 980 μL 1 X TE 버퍼 </td></tr><tr><td> 0.1 μg / ML </td><td> 1 μg / ML dsDNA 100 μL </td><td> 900 μL 1 X TE 버퍼 </td></tr><tr><td> 0.01 μg / ML </td><td> 0.1 μg / ML dsDNA 100 μL </td><td> 900 μL 1 X TE 버퍼 </td></tr><tr><td> 0.001 μg / ML </td><td> 0.01 μg / ML dsDNA 100 μL </td><td> 900 μL 1 X TE 버퍼 </td></tr></tbody></table></li></ol> 3. 표준 및 샘플 형광 측정 <ol><li> 피펫 150 그림 7 당 96 잘 접시의 우물 A1 - E1에 각 표준의 μL, 다음과 같은 테이블에 따라 : <table border="1"><tbody><tr><td> 잘 </td><td> 표준 </td></tr><tr><td> A1 </td><td> 1 μg / ML </td></tr><tr><td> B1 </td><td> 0.1 μg / ML </td></tr><tr><td> C1 </td><td> 0.01 μg / ML </td></tr><tr><td> D1 </td><td> 0.001 μg / ML </td></tr><tr><td> E1 </td><td> 1 X TE 버퍼 </td></tr></tbody></table></li><li> 그림 7에 표시된 H3에 우물 F1에 피펫 150 μL 알려지지 않은 샘플 </li><li> 다중 채널 피펫 사용을 위해 설계된 여물로 단계 1.2 준비 PicoGreen 솔루션을 하거라. 웰스 A1 - H3 150 μL PicoGreen 솔루션을 제공하는 멀티 채널 피펫을 사용합니다. 부드럽게 피펫과 혼합 용액 및 표준. </li><li> 플레이스 어두운 영역 판과 발전에 대한 반응에 대한 2-5분을 기다립니다. </li></ol> 4. 대표 결과 좋은 교정 곡선은 소프트웨어에 의해 보정 곡선의 표준과 계산 측정시 F - 7000 소프트웨어에서 제공하는 다양한 매개 변수를 사용하여 평가됩니다. 결정 계수는 측정 기준에 맞게의 선하심과 계산 보정 곡선을 나타냅니다. 가까이이 값이 &quot;1&quot;로되어 측정값 및 교정 곡선의 더 맞습니다. 값이 &quot;1&quot;에서 멀리 떨어진 경우, 기준, 다시 측정 다시 준비하거나 교정 곡선의 적합성은 변경해야합니다. 그림 11 PicoGreen 염료를 사용하여 dsDNA의 부량 위해 얻은 계산 결정 계수 = 0.99993와 교정 곡선의 예를 보여줍니다. <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="그림 11" /> 그림 11. dsDNA 보정 곡선의 예.

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Analytical Biochemistry. 249 (2), 228-238 (1997).</li></ol>

루이스 A. 모레노와 켄드라 L. 콕스이 문서에서 사용되는 악기를 생산하는 히타치 하이 테크놀로지 미국에 의해 고용됩니다.

조심하세요 샘플 준비 중에 pipetting뿐만 아니라 안정적이고 민감한 형광 분광 광도계를 사용하여 좋은 재현성 결과를 얻기 위해 필수적입니다. 이 테스트에 사용된 형광 분광 광도계의 경우, 그것은 microplate 리더 300 μL 최종 샘플 볼륨을 사용하여 제안합니다. 낮은 볼륨 감도를 감소하고, 큰 볼륨 우물 사이의 교차 오염의 원인이 될 수 있습니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Distilled H2O</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>P7589</td>
<td>&nbsp;</td>
<tr>
<td>Nalge Nunc Fluorescence Microplates p/n 237108</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>237108</td>
<td>&nbsp;</td>
<tr>
<td>12 Channel Eppendorf Micropipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>3516677</td>
<td>&nbsp;</td>
<tr>
<td>1000 &mu;L Eppendorf Pipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L Eppendorf Pipette </td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 mL serological pipet</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum foil</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette tips</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J1-0003</td>
<td>&nbsp;</td>
<tr>
<td>Microplate Reader Accessory for F-7000</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J0-0139</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

특히 작은 농도의 DNA,의 부량는 합성과 정화의 DNA 등 유전자 증폭 제품의 부량 등 진단 응용 프로그램 및 약물의 DNA 분자의 탐지와 같은 표준 분자 생물학의 assays 포함하여 생물 학적 응용 프로그램의 다양한 중요한 작업입니다 준비. 이 비디오를 동안 우리는 분자 프로브 퀀트 - 그것 PicoGreen 염색 시약 키트를 사용하여 dsDNA의 부량을 수행하는 마이크로 플레이트 리더 액세서리 갖춘 히타치 F - 7000 형광 분광 광도계의 능력을 보여주는 것입니다. F - 7000 형광 분광 광도계는 고감도 및 고속 측정을 제공합니다. 그것은 측정 형광, 발광과 인광 수있는 매우 유연한 시스템입니다. 몇 가지 측정 모드 파장 스캔, 시간 검색, 측광 및 3 - D 검사 측정을 포함하여, 사용할 수 있습니다. 분광 광도계는 microplate에 300 μL 샘플 볼륨을 사용할 때 플루오레신 50 picomoles의 범위에서 감도를 가지고 있으며, 60,000 NM / 분의 스캔 속도를 측정할 수있다. 또한 최대 농도의 넓은 범위의 보정 곡선의 사용을 허용 규모의 5 주문 다양한 동적 범위가 있습니다. 광학 시스템은 최대 에너지와 감성에 대한 모든 반사 광학을 사용합니다. 표준 파장 범위는 200-750 nm의이며, 옵션 근처 적외선 photomultipliers 중 하나를 사용할 때 900 nm의로 확장될 수 있습니다. 이 시스템은 옵션으로 외부 온도 조절 액체 순환 장치를 사용하여 섭씨 5-60도에서 플레이트 리더를위한 선택 온도 제어를 허용합니다. microplate 리더 96 잘 microplates의 사용을 허용하고, 반응 속도론 모드를 사용하는 경우 96 우물에 대한 측정 속도보다 60 초입니다. F - 7000 및 Microplate 리더를위한 소프트웨어 컨트롤도 매우 유연합니다. 샘플은 열 또는 행에 형식 중 하나에서 설정 수 있으며, 우물의 조합은 샘플 측정 선택할 수 있습니다. 이것은 microplate의 최적 활용을 허용합니다. 또한, 소프트웨어는 마이크로 Excel에서 만든 쉼표로 구분된 값에 저장된 판 샘플 구성 또는 &quot;CSV&quot;포맷을 가져올 수 있습니다. Microplate 측정 구성이 저장되고 편의를위한 소프트웨어 및 생산성 향상으로 리콜 수 있습니다. 데이터 결과는 표준 보고서 출력, 엑셀로, 또는 선택적 보고서 생성기 프로그램을 수 있습니다.

Watch this Scientific Journal Video about Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye at JoVE.com

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

분자 프로브 PicoGreen 염료와 microplate 리더 액세서리를 갖춘 히타치 F - 7000 형광 분광 광도계를 사용하여 dsDNA의 부량의 데모.

히타치 F - 7000 형광 분광 및 PicoGreen 염료를 사용하여 dsDNA의 부량

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

quantification-dsdna-using-hitachi-f-7000-fluorescence

Research

JoVE Journal

Biology

24.4K 조회수.  Hitachi High Technologies America. 분자 프로브 PicoGreen 염료와 microplate 리더 액세서리를 갖춘 히타치 F - 7000 형광 분광 광도계를 사용하여 dsDNA의 부량의 데모.

비디오: Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

NanoDrop 2000c 분광을 사용 Microvolume 단백질 농도 결정

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

연구

생물학

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

마이크로 볼륨 바이오 사양 나노 분광 광도계를 사용하여 핵산 및 단백질 농도 결정

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

파우더 샘플의 절대 양자 수율 측정

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

의 시각화 Caenorhabditis Elegans</emLipophilic 바이탈 염료, DiI를 사용하기> 표피 구조

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

관측 및 텔로미어의 정량 및 반복 시퀀스 사용하여 형광<em&gt; 현장에서</emPNA 프로브에와&gt; 하이브리드 (FISH)<em&gt; 예쁜 꼬마 선충</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

[18F]FDG 마이크로 PET/MR 이미징을 사용한 마우스의 갈색 및 베이지 지방 조직의 시각화 및 정량화

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

단세포 형광 화상 진찰을 사용하여 세균 간 경쟁의 정량화

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...