This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

혈액 샘플 실험은 관련 법률 및 제도적 지침에 따라 수행 하였다. 샘플은 인수 기관에서 임상 시험에서 처리했다 잔류 혈액 샘플을했다. 1. PT-AS 광 설치 참고 : 하나는 초기 PT-AS 설정에 빈 마이크로 모세관 튜브를 사용할 수 있습니다. <ol><li> 각각 200 및 330 μm의의 내부 및 외부 직경을 갖는 빈 마이크로 모세관 튜브 및 모세관 고정구에 큰 ~ 5 cm 이상의 길이를 탑재. 시판 섬유 일정 튜브 치구로서 사용할 수있다. </li><li> 안전하게 모세관을 조명하는 광원을 조사, 즉, 650 nm의 레이저 포인터를 고정. 프로브 빔이 모세관보다 커야한다. 각도주기적인 패턴을 관찰하는 모세관 뒤에 화면 (예 : 흰색 종이)를 놓습니다. </li><li> 검출부를 들어, 직접 scatt 캡처 웹캠 어떠한 렌즈를 제거패턴를 끄기. 프로브 빔 방향에 대해 25 ~ 35 °의 각도에서 모세관 뒤에 웹캠 배치. 모세관에 의해 생성 된 각주기 패턴 검출기 (도 1)에 의해 측정 될 수 있음을 보장한다. 이미지 센서가 위치 될 때, 이미지 센서의 중간 각도주기 패턴을 관찰한다. </li><li> 모세관을 조명 할 수있는 532 나노 PT의 여기 광원을 배치합니다. 만큼 PT 여기 광이 모세관 튜브 프로브 빔과 중첩 직접 검출기에 도달하지 않는 한, 임의의 각도로 PT 광원을 배치. 는 RI에 큰 변화를 초래으로 일반적으로 높은 광 출력을 이용하여 혈액 샘플의 PT 여기는 PT-AS 감도를 향상시킨다. <ol><li> 채용 된 PT 여기 광원에 가장 높은 광 출력을 사용한다. 또한, PT 여기 광이 모세관에 탐침 광을 중첩되도록. 태평양 표준시 여기 광의 빔의 크기를 사용하여배 이상의 광 프로브의 전체 프로브 체적을 가열. </li></ol></li><li> 532 nm의 광을 차단하고 단지 650 nm의 프로브 광을 측정하는 검출기 앞에 긴 패스 필터를 배치했다. </li><li> 모세관을 조사하기 전에 PT 여기 광의 경로에 광 초퍼를 설치한다. 광 초퍼는 PT의 여기 광 강도를 변조하는데 사용된다. </li></ol> 2. 혈액 샘플 준비 <ol><li> 에틸렌 디아민 테트라 아세트산 혈액 샘플링 튜브에 빈혈 상태에 신선한 전혈 6 ml의 그리기, 잘 샘플을 섞는다. 다른 처리가 필요하지 않습니다. </li><li> 혈액 응고를 방지하는 추출 24 시간 안에 PT-AS 센서를 이용하여 혈액 시료를 측정한다. </li></ol> 3. PT-AS 측정 프로토콜 <ol><li> 측정하기위한 혈액 샘플을 마이크로 모세관을로드. 혈액의 튜브에 배치하여 모세관 작용을 통해 혈액 모세관 채우기충분한. 측정에 필요한 최소 샘플 량은 모세관의 내경 및 프로브 빔의 크기에 의해 결정된다. <ol><li> 200 μm의 내경을 가진 튜브를 이용한다. 프로브 빔 사이즈 측정이&gt; 63 NL 샘플 부피로 수행 될 수 있음을 시사 대표적인 결과 2 mm였다. </li></ol></li><li> 고정구에있는 지정된 위치에있는 모세관 튜브를 탑재합니다. </li><li> 혈액이 장착 된 마이크로 모세관 튜브를 조명하기 위해 650 나노 프로브 레이저를 켭니다. 각도주기적인 패턴은 웹캠으로 관찰해야한다. </li><li> 튜브를 조명하기 위해 532 나노 PT 여기 레이저를 켭니다. </li><li> 2 Hz에서의 PT 여기 광의 강도를 변조하는 광 초퍼를 실행. 주 :이 운전 조건의 선택을위한 이론적 근거는 토론 및 Kim 등으로 설명된다. 12. <ol><li> 광 초퍼 모터 헤드 어셈블리 초퍼 휠 장착체계. </li><li> 헬기 컨트롤 박스의 전원을 켜고 변조 주파수를 설정 콘솔의 컨트롤 노브를 사용합니다. </li><li> 컨트롤 노브를 사용하여 헬기를 실행합니다. </li></ol></li><li> MPEG-4 (MP4) 형식으로 5 초 동안 웹캠을 통해 변동 산란 패턴을 기록합니다. </li></ol> 4. 신호 처리 주 : PT-AS 신호 처리 실험실 개발 MATLAB 코드를 사용하여 수행 하였다. <ol><li> 이미지를 추출 할 동영상 파일을로드합니다. 각 화상의 세로 방향의 화소 값의 평균을 계산하여 평균 산란 패턴을 얻었다 [대표 이미지도 2 (a)를 참조]도 2 (b, c)]. </li><li> 푸리에 평균 산란 패턴의 변형 평가하고 최고 공간 주파수에서의 위상을 계산한다. 모든 녹화 된 영상의 모든 프레임에 대해 이러한 작업을 수행합니다. </li><li> 모든 이미지로부터 획득 된 위상 값을 이용하여, 시간 상 플롯변동 [그림 2의 (d)]. 위상이 PT 변조 주파수 변동합니다. 푸리에 변조 주파수의 크기를 시간 영역에서의 위상 변동 변환 및 수득보십시오. 이 신호는 PT-AS 신호라고한다 [도 2의 (e)]. </li><li> 내로의 PT-AS 신호를 전환하여 혈액 샘플의 [헤모글로빈]을 측정 프로토콜 (5)에서 얻어진 검량선을 이용하여 헤모글로빈] 대응. </li></ol> 5. PT-AS 교정 <ol><li> (- 18g / DL 예컨대, 0)의 Hb] 균일 PT-AS 센서의 검출 범위에 분포 값을 갖는, 혈액 샘플을 준비한다. </li><li> 보정 전, 기준 혈액 분석기를 사용하여 샘플의 [헤모글로빈] 값을 정량화. 샘플의 PT-AS 신호를 측정한다. </li><li> experimenta의 교정 최소 제곱 적합 선형를 수행하여 PT-AS 신호 [헤모글로빈] 관련 곡선 [헤모글로빈] = A [PT-AS 신호] + B를 도출리터 결과. 표 1에 규정 된 작동 조건 [헤모글로빈] 및 PT-AS 신호 사이의 관계는 [헤모글로빈]였다 = 5.13 [PT-AS 신호] - 0.09. 선형 피팅을 수행하기 위해 MATLAB 코드를 사용합니다. </li></ol>

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No conflict of interest is declared.

PT-AS 센서는 처리되지 않은 혈액 샘플을 직접 [헤모글로빈] 측정 할 수있는 모든 광학 방법을 나타낸다. 상기 방법은 적혈구의 헤모글로빈 분자의 고유 PT 응답하여 혈중 [헤모글로빈] 정량화한다. 532 nm의 빛에 의해 조명 아래에서, 헤모글로빈 분자는 빛 에너지를 흡수하고 열을 생산하고 있습니다. 얻어진 온도 상승은 혈액 샘플의 RI를 변화시킨다. BSI의 높은 RI 감도는 혈액에서이 RI의 변화를 측정...

혈액 검사는 일반적으로 전체 인간의 건강을 평가하는 특정 질환에 관련 바이오 마커를 검출하기 위해 수행된다. 예를 들어, 혈중 콜레스테롤 농도는 밀접한 심혈관 질환 및 췌장염 관련되어 고지혈증의 기준으로서 역할을한다. 혈당 수준이 당뇨병 성 케톤 산증과 고혈당 삼투압 증후군과 관련된 합병증으로 혈당 내용은 자주 측정해야한다. 말라리아, 인간 면역 결핍 바이러스와 후천성 면역 결핍 증후군 같은 심각한 질병을 혈액 검사에 의해 진단하고, 적혈구, 혈소판을 포함한 혈액 성분의 정량 및 백혈구 췌장 및 신장 질환의 스크리닝을 가능하게한다. 헤모글로빈 (혈색소), 혈액의 중요한 구성 요소는 적혈구의 약 96 %를 구성하고, 사람의 장기에 산소를 수송한다. 질량 농도의 중요한 변경 ([혈색소])는 저를 나타낼 수있다tabolic 변경, 간담 질환,과, 신경 심혈관 및 내분비 장애 1. [혈색소 따라서 정기적으로 혈액 검사로 측정됩니다. 특히, 빈혈 환자, 투석 환자, 임산부에 강하게 중요한 작업이 같이 헤모글로빈]을 모니터링하는 것이 좋습니다. 다양한 [헤모글로빈] 검출 방법에 따라서 개발되었다. 헤모글로빈 시안화 방법 [헤모글로빈] 정량 가장 흔한 방법 중 하나는, 시안화 칼륨 (KCN)은 적혈구 (3)의 지질 이중층을 파괴하기 위해 사용한다. 540 nm의 주위 화학 나타낸다 높은 흡수에 의해 생성 된 시안 헤모글로빈; 따라서, [혈색소] 측정은 비색 분석을 통해 할 수있다. 이 방법은 널리 단순성 때문에 사용되지만 사용되는 화학 물질 (예, KCN 및 dimethyllaurylamine 산화물)은 인간과 환경에 독성이있다. 헤마토크릿 방식은 전체 혈액의 부피에 비하여 적혈구의 체적비를 측정원심 분리를 통해 매화; 그러나 비교적 큰 혈액량 (50-100 μL) (4)를 필요로한다. 측정법 방법의 측정은 [헤모글로빈] 정확히 어떤 화학 물질이없는,하지만 여러 파장과 큰 혈액량의 측정은 5,6 필요합니다. 유사하게, [헤모글로빈] 광학 측정을위한 여러 방법이 광 산란에 기초하여 검출 방법을 포함하는 제안되어 있지만, 그 측정 정밀도는 혈액 이론적 모델의 정확도에 강하게 의존한다. 이러한 한계를 극복하기 위해, 헤모글로빈의 광열 (PT) 효과에 기초하여 헤모글로빈] 검출 방법은 최근 7이 제안되어있다. 주로 산화철로 구성되어 혈색소는 532 nm에서 빛을 흡수하고 열 8-10로 빛 에너지를 변환합니다. 이 PT의 온도 상승은 혈액 시료의 굴절률 (RI)의 변화를 측정함으로써 광학적으로 검출 할 수있다. 임 등. 고용 스펙트럼 영역 빛 간섭 reflectometrY는 피 수용 실 (11) 내의 PT 광로 길이의 변화를 측정한다. 상기 방법은 무공해 직접 [헤모글로빈] 측정 가능하지만, 분광기의 사용과 간섭계 장치는 소형화를 저해 할 수있다. 최근 대안 [헤모글로빈] 검출 방법을 제시 장치 (12)의 소형화에 더 적합 광열 각도 광산란 (PT-AS) 센서를 불린다. PT-AS 센서 모세관 안에 혈액 샘플의 RI PT의 변화를 측정하는 백 산란 간섭계 (BSI)의 RI 높은 감도를 이용한다. BSI 다양한 솔루션 13-15의 RI를 측정하는 자유 용액 16 생화학 적 상호 작용을 모니터링하는 데 이용되어왔다. 태평양 표준시-AS 센서는 BSI와 같이 유사한 광학 장치를 사용하지만, 혈액 샘플에서 RI의 PT 증가를 측정하는 광열 여기 설정을 결합한 제품입니다. BSI에 상기 PT-AS 센서의 작동 원리는 다른 곳에서 상세하게 설명된다 <sup&gt; 12,15. PT-AS 센서는 넓은 감지 범위 (0.35-17.9 g / DL)을 통해 고감도 [혈색소] 측정을 입증하고 &lt;100 NL의 샘플 볼륨으로 운영 할 수있다. 혈액 샘플의 어떤 전처리가 불필요하며, 측정 시간은 5 초 ~하다. 여기서, 실험 장치 상세한 측정 프로토콜이 설명된다. 주제 PT-AS 결과 빈혈 환자의 혈액 샘플을 이용하여 제공되고, 그 결과는 PT-AS 센서의 정확도를 평가하기 위하여 혈액 분석 장치에서 그와 비교된다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>650nm laser pointer</td>
			<td>LASMAC</td>
			<td>LED-1</td>
			<td>Probe light</td>
		</tr>
		<tr>
			<td>Hollow round glass capillaries</td>
			<td>Cm Scientific</td>
			<td>CV2033</td>
			<td>Blood sample container</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C525</td>
			<td>CMOS optical sensor</td>
		</tr>
		<tr>
			<td>532-nm DPSS laser</td>
			<td>CNI Laser</td>
			<td>MGL-&#8546;-532</td>
			<td>Photothermal light source</td>
		</tr>
		<tr>
			<td>Optical chopper system</td>
			<td>Thorlabs</td>
			<td>MC2000-EC</td>
			<td>Optical chopper</td>
		</tr>
		<tr>
			<td>Plastic long-pass filter</td>
			<td>Edmund Optics</td>
			<td>#43-942</td>
			<td>To reject 532-nm PT excitation light</td>
		</tr>
		<tr>
			<td>Fiber clamp</td>
			<td>Thorlabs</td>
			<td>SM1F1-250</td>
			<td>Capillary tube fixture</td>
		</tr>
		<tr>
			<td>EDTA coated blood sampling tube</td>
			<td>Greiner Bio-One</td>
			<td>VACUETTE 454217</td>
			<td>Blood sampling &#38; anticoagulating</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Siemens AG</td>
			<td>ADVIA 2120i</td>
			<td>Reference hematology analyzer</td>
		</tr>
	</tbody>
</table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

광열 각도 광산란 (PT-AS)은 혈액의 헤모글로빈 농도 ([헤모글로빈])를 측정하는 신규 한 광학 방법이다. 헤모글로빈 분자의 고유 광열 응답에 기초하여, 센서는 고감도 [헤모글로빈]의 무농약 측정을 가능하게한다. [혈색소] 0.35의 범위에서 0.12 g / DL의 한계와 검색 기능 - 17.9 g / DL는 이전에 증명되었다. 이 방법은 용이하게 이러한 레이저 포인터 웹캠으로 저렴 가전 기기를 이용하여 구현 될 수있다. 혈액 용기와 같은 마이크로 모세관 튜브의 사용은 또한 나노 리터 규모 혈액량과 낮은 운전 비용으로 헤모글로빈 분석을 가능하게한다. 여기서, PT-AS 광 설치 및 신호 처리 절차에 대한 자세한 내용은되게됩니다. 실험 프로토콜 및 빈혈 상태에서 혈액 샘플 대표적인 결과 ([헤모글로빈 = 5.3, 7.5 및 9.9 g / DL)도 제공되고, 측정은 그 이리저리 비교엄마의 혈액 분석기. 구현 및 운영에 단순 임상 실험실 및 자원 제한 설정에서 넓은 채택을 사용하도록 설정해야합니다.

헤모글로빈 분석은 PT-AS 센서를 이용하여 수행하였고, 그 측정은 혈액 분석 장치에서 비교 하였다. 실험은 1.4 W / cm 2 초, 5, PT 변조 2 Hz의 주파수 및 측정 시간 PT 여기 광 강도로 하였다. 표 1은 실험 조건을 요약한다. 프로브 및 PT 여기 광의 빔의 크기는 각각 5.5 및 2mm이었다. 웹캠 30 fps의 프레임 율로 이미지를 기록했다. 측정을 위해, 세 가지의 Hb 농도 ?...

Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

광열 각도 광산란와 신속하고 화학 무료 헤모글로빈 분석

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

a-rapid-chemical-free-hemoglobin-assay-with-photothermal-angular

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JoVE Journal

Bioengineering

9.6K 조회수.  Yonsei University. A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

비디오: A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

미세 유동 프로브와 라이브 셀 레이어의 빠른 빼기 패터닝

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

연구

생체공학

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

세포 배양 응용 프로그램에 대한 히드로 겔의 빛 매개 형성 및 패터닝

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

바이오 메디컬 응용 프로그램에 대한 Bioinspired 나노 수산화 인회석의 빠른 혼합 준비

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

키네 성장 시험을위한 온도, 빛 및 pH 모니터링을 갖춘 벤치 규모의 조류 광합성 생물 반응기의 건설 및 설치

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

성인 골 수 유래 조 혈 줄기 세포로 세포 공학 자기 대상으로 함께 있도록 예측에 관한 전송을 위한 프로토콜

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

아메바를 사용 하 여 빠른 이미지 기반 세균 독성 분석 결과

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

무세포 시스템을 사용한 유전 부분의 신속한 특성 분석

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

미생물 화학 및 단백질 생산을 위한 광 조절 발효

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

생체 내 결합 된 이광자 형광 및 자극 된 라만 산란 현미경을 사용한 생물학적 조직 이미징

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

RNA 편집의 신속한 검출을 위한 비시퀀싱 접근법

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...