1 부 : BioFlux 판의 microfluidic 채널의 준비. <ol><li> 흐름 세포 실험을 실행하기 전에, 하나는 관심의 단백질 코팅 microfluidic 채널을 준비해야합니다. 이 경우에는 콜라겐은 내가이 사용됩니다. 각 채널은 잘 입구와 콘센트가 있습니다. 이 접시는 입구가 잘 잘 채널과 우물이 오른쪽에있는 콘센트를주고 왼쪽입니다. </li><li> 0.02M 초산에 200μg/ml 농도 콜라겐 I (5mg/ml 주식) 코팅을 희석. 하나는 사용되는 각 채널에 대해 20μl가 필요합니다. micropipette 팁과 부드러운 triteration하여 섞는다. </li><li> 사용되는 각 해당 채널에 코팅 20 μl를 추가합니다. 하나는 잘 micropipette를 사용하여 관심있는 microfluidic 채널을 먹이 콘센트의 내부 펀치로 액체를 분배합니다. 거품을 도입하지 마십시오, 피펫의 공기를 밀어하지 않습니다. 콜라겐 컨트롤을위한 콜라겐없이 하나의 채널을 (접착 및 집합에 대한 부정적인 컨트롤)을 포함시키십시오. </li><li> 4 개의 나사 그리고 그들이 모두 설정되면 완전히 조이 토크 드라이버를 사용 강화 첫 번째 손가락으로 접시에 인터페이스를 연결합니다. 그것이 최대 죄어져 있음에 도달하면 토크 드라이버를 클릭합니다. </li><li> BioFlux 소프트웨어에서 수동 모드를 사용하여 콘센트에 잘 2dyn/cm -에서 2 관심의 채널에 재관류를 적용할 수 있습니다. 여기 하나는 액체로 가득하는 반대 안쪽 펀치에 대한 감시해야합니다. 이것은 몇 분 정도 소요됩니다. 하나는 하나 이것은 현미경의 낮은 전력을 사용하는 목적 (4X)과 잘하거나 접시를 들고 아래에서 유입 우물을 조사하여 입구를 찾는가 발생할 볼 수 있습니다. 하나는 먼저 천천히 안쪽 펀치를 채우고 액체의 작은 구슬이 나타납니다. 입구 안쪽 펀치가 가득되면, 소프트웨어의 중지를 추진하여 한번에 재관류를 중지합니다. </li><li> 한 시간 만이라도 실온에서 접시를 품어. </li><li> 인터페이스를 제거하고 잘 콘센트에 PBS (플러스 칼슘 2 + / MG 2 +) 1 ML를 추가합니다. 2dyn/cm 2 잘 콘센트에서 재관류를 시작합니다. 10 분 동안 재관류를 계속합니다. 재관류를 중지하고 인터페이스를 제거합니다. </li><li> 입구와 출구의 우물 모두에서 초과 PBS를 제거 - 내부 펀치를 채우고 액체를 제거하지 마십시오. 차단 솔루션 (0.5 % PBS의 V / V BSA (플러스 칼슘 2 + / MG 2 이상)을)를 사용하여 채널을 차단합니다. 사용할 수 있도록 잘 각 콘센트에 차단 솔루션 1 ML을 추가하고 2dyn/cm 2 잘 콘센트에서 perfuse. 10 분 동안 재관류를 계속합니다. 재관류를 중지하고 인터페이스를 제거합니다. 이 단계까지 준비 채널이 하루 동안 사용할 수 있습니다. </li><li> 혈액을 추가하기 전에, 안쪽 펀치를 제외하고 채널의 양쪽에있는 액체를 제거합니다. </li></ol> 2 부. GPIIb / IIIa 억제 항체와 함께 혈액을 준비. <ol><li> 콜라겐이 채널에 잠복기이지만, 하나는 전체 혈액을 준비합니다. 하나는 인간의 혈액을 다룰 때 그 / 그녀의 연구소에 의해 결정 biosafety 규정을 따라야합니다. </li><li> 나트륨 구연 산염의 항응고제로 그려 신선한 인간의 피를 (금식 개인에서) 컬렉션 3 시간 이내에 사용해야합니다. </li><li> AM 4μM calcein을 추가하여 혈액을 준비합니다. DMSO의 AM calcein의 4mm짜리 주식을 준비하고 1천분의 1 V /의 희석에 혈액에 추가 V. 부드러운 역전로 섞는다. </li><li> 1.5mL microtubes - 1 calcein의 ML 10에 혈액 라벨 AM 하는걸. 원하는 희석 (IgG의 분자량은 150kDa ~입니다)에서 각 GPIIb / IIIa 억제제 항체를 추가합니다. 예제 희석 시리즈 1200nM에서 9nM하는 것입니다 그리고 항체 컨트롤도 및 관련없는 항체 컨트롤을 포함하지 않습니다. 억제를위한 훌륭한 긍정적인 제어 20ug/ml의 농도에서 (압식시맙, 엘리 릴리) ReoPro 것입니다. 온화한 역전의 혼합. </li><li> 1 시간 동안 실온에서 알을 품다. 부드러운 역전로 10 분마다 섞는다. </li></ol> 파트 3 : BF1000 워크 스테이션에서 유동 세포 실험을 실행. <ol><li> 첫째, 하나는 BioFlux 제어 모듈에 자동 프로토콜을 설정해야합니다. 콜라겐을 위해서라면, 하나는 잘 콘센트에서 10 분 동안 10dyn/cm 2 실행하는 프로토콜을 설정해야합니다. 이 시점에서 하나도 BF1000 워크 스테이션을 사용하여 데이터 수집 매개 변수를 설정해야합니다, 채널 위치를 포함하여, FITC 파장 및 시간 - 저속 정보를 캡처합니다. 그것은 총 10 분 기간 동안 채널당 10X 목표 30 초마다를 사용하여 볼 3 분야를 캡처하는 것이 좋습니다. </li><li> 별도의 준비 채널로 각 조건의 준비 피가 500ul 빠르게 작업, 장소. 콜라겐 그리고 차단 하나 코팅하는 채널로 아무 항체 제어 혈액 플레이스 없습니다. </li><li> 접시에 인터페이스를 배치합니다. </li><li> BF1000 워크 스테이션 현미경에서 접시를 놓습니다. 인터페이스에 클램프. </li><li> 플레이트 부하 조정을 수행합니다. 또한, 하나 잘 들어있는 혈액으로 무대를 이동합니다. 설정 FTIC 이미지 캡쳐 매개 변수 (노출 시간, 이득 등 ...). </li><li> BioFlux 몬티지 소프트웨어에서 동시에 유동 및 수집을 시작하는 인수를 시작합니다. </li><li> BF1000 워크 스테이션에서 접시를 제거, 기관 지침에 따라 접시의 처분. </li></ol> 4 부 : 대표 결과. 여기 microfluidic 채널 혈소판 점착 및 집계를위한 프로토콜이 제시되었다. 혈소판 응집 억제제 치료는 anti-GPIIb/IIIa 또한 프로토콜에 포함되었다. BioFlux 시스템의 콜라겐 코팅 microfluidic 채널을 사용하여, 하나는 치료 제어 혈액 샘플과 uncoated 채널없이 혈전 형성과 함께 시간이 지남에 따라 적극적인 혈전 형성을 볼 것으로 예상한다. 한 최근 수행한 실험에서, 제어 조건 집계의 평균 크기는 2000μm이되었습니다. 그림 1. <img src="/files/ftp_upload/1644/1644fig1.jpg" alt="그림 1" /> 활성 GPIIb / IIIa는 혈소판 - 상호 작용과 혈소판 집합 안정화 1 강력한 중보자입니다. 콜라겐에 부착이 응답 2 이끌어내는 수있는 GPIIb / IIIa 복잡한을 활성화한다. 전단 노출 이전에 1 시간 anti-GPIIb/IIIa과 부화 후, 하나는 thrombi의 크기의 감소뿐만 아니라 혈전 형성의 빈도 감소를 관찰할 것으로 기대한다. 선량에 의존 반응은 일반적으로 10dyn/cm 2 볼 수 있습니다. 그림 2. <img src="/files/ftp_upload/1644/1644fig2.jpg" alt="그림 2" /> 이 특정 억제제에 대한 IC 50 값은 10 dyn / cm 2 17nM되었습니다. 노 항체 컨트롤에 비해 최대 억제 (이 기증자에 대한) 10 dyn / cm 2 11%되었다. 여기에 방법이 특정 세포외 기질 단백질과 혈소판 집합의 특정 억제제를 사용하여 제시하는 동안, 프로토콜은 다른 코팅 다른 세포 종류와 세포 유착 assays 다른 억제제로 확장됩니다. 이러한 실험의 성공의 핵심 요소는 모든 시약에 대한 microfluidic 채널, 올바른 diluents의 모든 시약의 정확한 희석하고, 적절한 배양 조건에서 거품의 소지를 없애기 있습니다.

<ol>
	<li>Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+complexity+of+platelet+aggregation.">The growing complexity of platelet aggregation.</a> Blood. 109, 5087-5095 (2007).</li><li>Nakamura, T., Kambayashi, J., Okuma, M., Tandon, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+GP+IIb-IIIa+complex+induced+by+platelet+adhesion+to+collagen+is+mediated+by+both+alpha2beta1+integrin+and+GP.">Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP.</a> VI. J Biol Chem. 274, 11897-11903 (1999).</li></ol>

기여 저자 Fluxion Biosciences의 직원입니다.

여기에 방법이 특정 세포외 기질 단백질과 혈소판 집합의 특정 억제제를 사용하여 제시하는 동안, 프로토콜은 다른 코팅 다른 세포 종류와 세포 유착 assays 다른 억제제로 확장됩니다. 이러한 실험의 성공의 핵심 요소는 모든 시약에 대한 microfluidic 채널, 올바른 diluents의 모든 시약의 정확한 희석하고, 적절한 배양 조건에서 거품의 소지를 없애기 있습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>collagen I, Bovine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A1064401</td>
<td>other sources of collagen I can be used</td>
</tr>
<tr>
<td>PBS plus Ca/Mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14040-117</td>
<td>&nbsp;</td>
<tr>
<td>Bovine serum albumin (BSA)</td>
<td>Reagent</td>
<td>EMD</td>
<td>EM-2930 Bottom of Form</td>
<td>From VWR</td>
</tr>
<tr>
<td>calcein AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C1430</td>
<td>Calcein AM from BD can also be used</td>
</tr>
<tr>
<td>BioFlux 1000 System</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>Contact Company</td>
<td>&nbsp;</td>
<tr>
<td>BioFlux Plate</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>900013</td>
<td>&nbsp;</td>
<tr>
<td>antiGPIIb/IIIa</td>
<td>Reagent</td>
<td>Abcam</td>
<td>ab33407</td>
<td>an alternative # is ab15021</td>
</tr>
<tr>
<td>ReoPro</td>
<td>Reagent</td>
<td>Eli Lilly</td>
<td>&nbsp;</td>
<td>by Rx only</td>
</tr>		</tbody>
		</table>
		</div>

platelet adhesion and aggregation under flow using microfluidic flow cells

혈소판 응집은 내피 아래의 세포외 기질이 노출되었습니다 혈관 손상에 대한 응답으로 발생합니다. 혈소판 유착 계단식는 전단 흐름의 존재에서 일어나는, 요인은 종래의 (정적) 잘 철판 assays에서 차지하는 없습니다. 이 문서의 생리 전단 흐름 조건을 에뮬레이트하는 microfluidic 잘 플레이트 형식을 이용한 혈소판 - 집계 분석에 대한 보고서. 세포외 단백질, 콜라겐 I 또는 폰 Willebrand 인자는 공기 펌프와 적극적인 재관류를 사용하여 microfluidic 채널 내에 입금됩니다. 매트릭스 단백질은 다음 버퍼로 세탁 및 혈소판 상호 작용에 대한 microfluidic 채널을 준비하는 차단됩니다. 형광 염료로 분류 전체 혈액은 혈소판 활성화 및 집계를 달성하기 위해 다양한 유량에서 채널을 통해 perfused입니다. 혈소판 응집 억제제는 IC50의 선량 응답 데이터를 생성하는 흐름 세포 실험 이전에 추가할 수 있습니다.

Watch this Scientific Journal Video about Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells at JoVE.com

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

혈소판 유착 계단식는 전단 흐름의 존재에서 일어나는, 요인은 종래의 (정적) 잘 철판 assays에서 차지하는 없습니다. 이 문서의 생리 전단 흐름 조건을 에뮬레이트하는 microfluidic 잘 플레이트 형식을 이용한 혈소판 - 집계 분석에 대한 보고서.

Microfluidic 흐름 전지를 사용하여 흐름에서 혈소판 접착 및 집계

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

platelet-adhesion-aggregation-under-flow-using-microfluidic-flow

Research

JoVE Journal

Biology

18.1K 조회수.  Fluxion Biosciences, Inc.. 혈소판 유착 계단식는 전단 흐름의 존재에서 일어나는, 요인은 종래의 (정적) 잘 철판 assays에서 차지하는 없습니다. 이 문서의 생리 전단 흐름 조건을 에뮬레이트하는 microfluidic 잘 플레이트 형식을 이용한 혈소판 - 집계 분석에 대한 보고서.

비디오: Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

마우스 Meiotic 셀 유동세포계측법의 정제

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

연구

생물학

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

셀 추적 염료를 사용하여 전지 부문 모니터링에 최적화 된 착색 및 확산 모델링 방법

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

라이브 셀의 단백질 응집체의 4D 영상

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

프로토 타입 소형 흐름 사이토와 동반자 미세 유체 혼합 기술의 감소 - 중력 환경 하드웨어 데모

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED 열 흐름 - 유동 세포 계측법으로 Optogenetics 결합

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

스펙트럼 유동 세포 계측법에 의해 고체 조직에서 분리 한 세포 현탁액의 분석

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

흐름하에있는 혈소판 탈식 및 분비의 라이브 셀 이미징

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

자기 적고를 이용한 세포 밀도 및 항원 적 특성의 정량화

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

인간 1차 출처에서 인간 MK의 면역페노티핑 및 세포 분류 또는 헤마토포이틱 전조자로부터 시험관내 분화

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

다운스트림 적용을 위한 별개의 지방 저장소에서 혈관 내피 세포의 분리 및 식별

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...