We would like to thank Henriette Mair-Meijers for invaluable attention to detail and hours devoted to processing the blood collections. We are grateful for the graphic design expertise of Natalie Jameson Kiesling. We also appreciate the approval of the manufacturers (Qiagen (Valencia, CA), BD Biosciences (San Jose, CA), Life Technologies (Grand Island, NY)) mentioned herein to publish the use of their products as described. Funding for this work was generously provided by the National Institutes of Health award numbers DA022720, RC1MH088283, and DA022720-05-S1.

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저자는 그들이 더 경쟁 이익이 없다고 선언합니다.

우리는 성공적으로 디트로이트 환경 건강 연구에 1,600 개 이상의 전체 혈액 샘플을 처리하기 위해 적용된 간소화 된 프로토콜을 설명했다. 이들 기술의 대부분은 기존 문헌에서 이용할 수 있지만, 각 단계 사이에 정확하게 타이밍 변화를 포함하여 단계별 편집, 성공적 하류 광범위한 응용으로 생물학적 시료의 종류를 생산 최적화 효율적인 프로토콜이 반영 DNA 메틸화, mRNA 발현과 면역 학적 분석?...

여러 연구와 정신 (또는 다른) 1-4 질병없이 개인간 혈액 유전자 발현, DNA 메틸화 및 세포 서브셋의 차이를 특징으로하고있다. 이러한 연구는, 그러나, 질병 - 관련 차이 때문에 환자를 치료하고자하는 질병에 대해 일반적으로 더 심한 특성을 확대 할 수있는 임상에서 얻어졌다. 때문에 &quot;오 믹스&quot;접근 방법의 발전으로, 지난 십 질병 유병률의 인구 기반의 추정치를 제공하기 위해, 지역 사회 및 / 또는 역학적 설정 5-7에서 생물학적 샘플을 얻는에 대한 관심의 폭발과의 폭 넓은 사진을 보았다 이러한 정신적 / 육체적 질병의 환경 적 결정. 이에 대한 주요 과제는 수집 된 표본의 신속한 처리에 대한 요구이다. 단핵 세포, frequentl 아르 키 면역 시스템 구성 요소들의 열화Y는 개인의 건강, 수집 8-10의 2 시간 후 복구에 상당한 감소와 혈액 무승부를 즉시 시작 평가하는 데 사용됩니다. 이 문제를 해결하기 위해, 우리는 인간의 전체 혈액의 여러 구성 요소가 동시에 대도시 지역에 사는 주제의 가정에서 얻어진 샘플로부터 고립되는 최적화 된 프로토콜을 제시한다. 프로토콜은 우리의 컴파일 및 미래의 기술이 더 분리를 허용 이벤트에있는 모든 &quot;추가&quot;분수의 저장을 포함하여 현재 기술의 변경에 기반 / 분석한다. 다른 방법 또는 키트가 여기에 설명 된 각각의 방법, 그 윤곽의 장소에서 이용 될 수 있지만 높은 처리량 방법으로 샘플을 처리하기위한 안정적이고 효율적인 방법으로 증명되었다. 양질의 분획 (PBMC를는, DNA는 혈청 및 RNA) 신선한 혈액의 2 일 (도 1 내에서 이용 가능하다 (2)의 수집 시간과 모든 분석 준비 시험편 내에 제조 할 수있다). 이 프로토콜은 지역 사회 주거, 디트로이트 환경 건강 연구에서 테스트 디트로이트시의 성인 거주자에서 수집 된 샘플의 효율적인 처리를 가능하게하기 위해 개발되었다 (DNHS을, DA022720, RC1MH088283, DA022720-05-S1)을, 인구​​ 기반 외상 후 스트레스 장애 (PTSD) 및 기타 정신 질환의 사회적, 생물학적 결정 요인 연구. 디트로이트에서 외상 후 스트레스 장애의 유병률은 두 번 전국 평균 11, 12 이상입니다. 이 집단에서 PTSD의 생물학적 결정을 확인하는 것은이 도시의 인구 및 기타 위험이 높은 집단 (예를 들어, 군 참전 용사를 반환) 모두에서 장애 그 고통을 돕기 위해 적절한 약물 및 / 또는인지 행동 개입을 개발하는 데 도움이 될 수 있습니다. 이전에 디트로이트, 미시간 웨인 주립 대학에있는 우리의 실험은, 다양한에서 파생 된 신선한 조직 샘플을 처리하는 우리의 전문 지식을 기반으로 처리를 위해 선정되었다소스, 필요성이 컬렉션의 2 시간 이내에 샘플을 처리하기 시작하고, 수집 사이트에 우리의 근접. 손이 독특한 기회, 우리의 목표는 각 시료로부터 DNA, RNA, 혈청 및 말초 혈액 단핵 세포 (PBMC)의 최대 수율 (시편 컬렉션 5 파도 위에 N = 1,639 샘플의 합계)에 대한 처리를 최적화하는 것이다. 여기에 설명 된 절차에 따라서, 마이크로 어레이, 성적인, 실시간 RT-PCR을 포함하여 하류 다양한 응용을위한 재료 (평균 수율은 표 1 참조)을 출발 제조, 비 임상 환경에서 동시에 수행하고, 분석 유세포 수있다. <img alt="그림 1" src="/files/ftp_upload/52227/52227fig1highres.jpg" width="700" /> 그림 1. 전체 작업 흐름. 여기에 도시 된 전체적인 공정은 동의 partic 식별로부터 혈액 표본을 얻는 물류를 포함혈액에 ipants 자체를 그립니다. 고품질, 분획 (말초 혈액 단핵 세포; PBMC를, DNA, 혈청, 및 RNA) 신선한 전혈의 컬렉션의 2 시간 내에 제조 될 수 있고, 모든 분석 시료 준비 2 일 내에 사용할 수있다. 시료는 즉시 시험 될 수없는 경우 또한,이 방법에 의해 제조 된 분획은 장기간 저장에 적합하다. 여기에 설명 된 전체 타임 라인은 하루 (~ 5 시간의 합계)에 완료 될 수 있습니다. 그러나, 이와 같은 일이 특히 상당한 경험과 기술을 가진 단일 기술자 매우 노동 집약적 일 것이다. 따라서, 우리는 적어도 두 기술자 사이에 1 일의 절차를 나누고 일 2. RNA 처리를 완료 추천 <a href="https://www.jove.com/files/ftp_upload/52227/52227fig1highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>QIAamp DNA Blood Mini Kit</td>
			<td>Qiagen</td>
			<td>51104</td>
			<td>Day 1: DNA isolation</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5493-1L</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>5 &#190;&#8221; Pasteur pipets</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), heat inactivated</td>
			<td>Life Technologies</td>
			<td>10082147</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418-500ml</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640, liquid</td>
			<td>Invitrogen</td>
			<td>11875119</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>0.4% trypan blue stain&#160;</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter or cell counting device such as a microscope and hemocytometer</td>
			<td>Invitrogen</td>
			<td>C10281</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>LeukoLOCK Fractionation &#38; Stabilization Kit&#160;</td>
			<td>Ambion</td>
			<td>1933</td>
			<td>Day 1: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>25G x 5/8 in. needles</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td>Day 1: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Syringes (5ml)</td>
			<td>Becton Dickinson</td>
			<td>309646</td>
			<td>Days 1 and 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Denaturing Lysis Solution&#160;</td>
			<td>Ambion</td>
			<td>8540G</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>5M NaCl</td>
			<td>Life Technologies</td>
			<td>24740011</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>TRI Reagent&#160;</td>
			<td>Ambion</td>
			<td>9738</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Bromo-3-chloro-propane (BCP)</td>
			<td>Sigma</td>
			<td>B-9673</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>spin cartridges&#160;</td>
			<td>Ambion</td>
			<td>10051G</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>0.1mM EDTA</td>
			<td>Ambion</td>
			<td>9912</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>DNA-free Kit</td>
			<td>Ambion</td>
			<td>AM1960</td>
			<td>Day 2: DNase treament</td>
		</tr>
		<tr>
			<td>RNA 6000 Ladder&#160;</td>
			<td>Agilent</td>
			<td>5067-1529</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>RNA 6000 Nano Series II Kit&#160;</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>RNaseZAP</td>
			<td>Ambion</td>
			<td>AM8782</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>Ethanol &#62;99%</td>
			<td>Sigma</td>
			<td>E7023-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol &#62;99%&#160;</td>
			<td>Sigma</td>
			<td>I9516-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free ultra pure water&#160;</td>
			<td>Invitrogen</td>
			<td>9938</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (nuclease-free)</td>
			<td>Eppendorf</td>
			<td>22491253</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter (serological)</td>
			<td>Eppendorf</td>
			<td>2223020-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetters (for volumes under 1ml)</td>
			<td>Eppendorf</td>
			<td>3120000-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes (serological)</td>
			<td>Fisher</td>
			<td>13-678-27E</td>
			<td></td>
		</tr>
		<tr>
			<td>Controlled rate freezing containers</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes (to hold 2ml and 5ml cryovials and 1.5ml microcentrifuge tubes)</td>
			<td>Fisher</td>
			<td>03-395-464</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tube rack</td>
			<td>Thermo Scientific</td>
			<td>14-804-134</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene tubes&#160;</td>
			<td>Fisher</td>
			<td>14-959-49D</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml and 0.65 microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>07-200-534 and 07-200-185</td>
			<td></td>
		</tr>
		<tr>
			<td>2ml and 5ml cryovials</td>
			<td>Fisher</td>
			<td>10-500-26 and 10-269-88F&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>8ml CPT vacutainer</td>
			<td>BD Biosciences</td>
			<td>362761</td>
			<td>2 tubes</td>
		</tr>
		<tr>
			<td>6ml K2 EDTA vacutainer</td>
			<td>BD Biosciences</td>
			<td>367863</td>
			<td>2 tubes</td>
		</tr>
		<tr>
			<td>8.5ml SST vacutainer</td>
			<td>BD Biosciences</td>
			<td>367988</td>
			<td>1 tube</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry bath incubator with heating block for microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>11-715-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration/vacuum system for use within the cell culture hood</td>
			<td>Fisher</td>
			<td>01-257-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed-angle rotor for microcentrifuge tubes with aerosol-tight lid</td>
			<td>Eppendorf</td>
			<td>22637002</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge with a swing-bucket rotor and aerosol-tight caps for 16 x 125mm vacutainers and 15ml polypropylene tubes&#160;</td>
			<td>Eppendorf</td>
			<td>22628157</td>
			<td>2, one does not need to be refrigerated</td>
		</tr>
		<tr>
			<td>Nanodrop 2000 (recommended for accuracy of small volumes) or other spectrophotometric device</td>
			<td>Fisher</td>
			<td>13-400-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen tank</td>
			<td>Thermo Scientific</td>
			<td>11-676-56</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#730;C freezer</td>
			<td>Thermo Scientific</td>
			<td>992RAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharps container</td>
			<td>Fisher</td>
			<td>22-037-970</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological waste container</td>
			<td>Thermo Scientific</td>
			<td>1223P52</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Level 2&#160; certified cell culture hood</td>
			<td>Thermo Scientific</td>
			<td>13-261-315</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid fractionation and isolation of whole blood components in samples obtained from a community-based setting

비 임상 설정에서 수집 및 전체 혈액 샘플의 처리와 기존 조건없이 모두 지역 사회에 거주하는 개인을 평가하는 독특한 기회를 제공합니다. 이들 샘플의 신속한 처리가 주요 세포 성분의 열화를 방지하는 것이 필수적이다. 동시에 말초 혈액 단핵 세포 (PBMC), DNA에 대한 방법이 여기에 포함된다, RNA 및 처리와 대도시 지역에 걸쳐 참가자를 동의의 가정에서 수행 한 혈액 추첨에서 혈청 분리, 컬렉션의 2 시간 이내에 시작했다. 우리는 1,600 혈액 표본이어서 성공적인 DNA 메틸화, 유전자형, 유전자 발현에 이용하고 유동 세포 계측법 분석 한 항복 일관된 고품질의 재료를 처리하는 이러한 기술을 이용했다. 사용되는 방법 중 일부는 표준; 설명 된 방식으로 조합 그러나, 그들은 인구 문제 및 / 또는 참가자를 커뮤니티의 샘플의 효율적인 처리를 가능하게일반적으로 임상에서 평가되지 않는다 기반 연구. 따라서,이 프로토콜은 일반적인 인구의 대표적인 (후속 데이터) 샘플들을 획득 할 수있는 잠재력을 갖는다.

이것은 전체적인 절차 유전자 마이크로 어레이와 RT-PCR 분석을 통해 발현 후성 변형을 검출하고, 셀 집합의 변화를 포함 하류 다수의 애플리케이션에서 분석을위한 높은 품질의 재료를 생성하는 것이 필수적이다. 표 1은 물질의 평균 수율 및 품질을 나타내는 각 프로세스에서. 3 백혈구 RNA 분리 및 필터 처리 방법의 품질의 출력의 예를 그림. 도 3의 좌측 이미?...

Watch this Scientific Journal Video about Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting at JoVE.com

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

지역 사회 기반의 설정에서 획득 샘플의 신속한 분별 및 전체 혈액 성분의 분리

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

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Research

JoVE Journal

Medicine

15.9K 조회수.  University of Illinois at Urbana-Champaign. We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

비디오: Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

연구

의학

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

α-테스트 : 전체 타액을 사용하여 빠른 셀 프리 CD4의 열거

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

신선한 수술 샘플에서 분리 및 인간의 심실 심근 기능 특성

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

설정 - 업<em&gt; 체외</em쥐 뇌 혈관 벽의&gt; 모델 (BBB)​​ : BBB 불 침투성 및 수용체 매개 운송에 초점

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

컬렉션, 격리, 인간 자궁 경부 샘플의 유세포 분석

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

인간 전혈에서 실시간 세포 독성 분석 실험

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

경피적 바늘 생검에 의해 획득 골격근 조직에서 고립 된 미토콘드리아의 준비 및 호흡계 평가

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

전체 조직과 조직 검사에서 인간 상피 Enteroids과 Colonoids 설립

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.