이 연구는 NIH 연구 기금 (DK090868과 TI의 GM092930)에 의해 지원되었다. 갇힌 rapamycin 유사체에 대한 출원 특허가있다.

1. 플라스미드 DNA의 Transfection <ol><li> 37.5 μl DH 2 O.로 형광 단백질로 태그 : 플라스미드 DNAs, 0.5 μg 멤브레인-닿는 곳에 FRB (이하 LDR로 지칭)와 0.5 μg FKBP-Tiam1을 (RAC 활성 T-세포 림프종의 침략과 전이 - 유도 단백질 1) 추가 1 μl FuGENE HD를 추가합니다. </li><li> 20 분 동안 상온에서 소용돌이와 부화. 한편, 단계 1.3-1.8로 진행합니다. </li><li> 50 μl 폴리-D-라이신과 8 아니라 챔버의 각 잘 씻으십시오. </li><li> DH 2 O를 사용하여 각 우물의 중심을 씻으 </li><li> 80~85% 합류 NIH-3T3 세포를 Trypsinize 10 ML 문화 매체 (10 % FBS가있는 DMEM)에 희석. </li><li> 정지 375 μl를 제거하고 3 분 동안 1,750 rpm으로 원심 분리기. </li><li> 펠렛을 방해하지 않도록주의되는 미디어를 기음. </li><li> DMEM w / 10 % FBS와 resuspend의 750 μl를 추가합니다. </li><li> DH 2 O / 유전자 / FuGENE의 HD 솔루션에 세포 현탁액을 넣고 가볍게 섞는다. </li><li> 250 추가각도에 대한 세포 / DNA를 정지 μl (3 우물까지 작성됩니다.) </li><li> 37 transfected 세포를 품어 ° C와 15-18 시간에 대한 5퍼센트 CO 2. </li></ol> 2. cRb-솔루션의 작성 <ol><li> 합성 갇힌 rapamycin-비오틴의 adduct (cRb). 자세한 내용은 &quot;cRb의 합성 구조&quot;를 참조하십시오. 간단히, caging 및 비오틴 moieties는 별도로 준비가되어 있습니다. caging 잔기 그때 rapamycin (LC 실험실)에 복합이며, 제품은 이후 cRb를 살만큼 클릭 반응 7 수단을 이용하여 비오틴 잔기에 결합. </li><li> DMSO의 cRb 중 1 μm의 주식 솔루션을 준비합니다. </li><li> 0.5 ML Eppendorf 튜브에 PBS 250 μl에 1mg 아비딘를 해산. </li><li> 녹아 아비딘에 cRb 주식 솔루션 1 μl를 추가합니다. 반전을 여러 번하여 섞는다. </li><li> cRb - 아비딘 켤레축을 (cRb-양보하는 15 분 동안 상온에서 부화) 솔루션입니다. </li><li> 언바운드 작은 분자를 제외하고 정화 cRb - 크기 - 제외 컬럼을 사용합니다. </li></ol> 3. 현미경과 Subcellular 레벨 라이트 일루미 네이션 <ol><li> 세럼은 12 시간 동안 FBS없이 DMEM에 transfected 세포를 굶어. </li><li> 부드럽게 세포 밖 미디어를 대기음하고 준비한 400 nm의 cRb - 솔루션 (단계 2.6에서) 250 μl를 추가합니다. </li><li> 빛 치료 전후 subcellular 해상도 세포를 생활에 단백질 역학을 시각화하기 위해 회전 디스크 현미경은 (그림 2A) 사용되었다. 자외선 조명은 에피 형광 라이트 (그림 2B)와 자외선 LED가 라이트 (365 나노미터)의 광학 장치를 결합하여 달성되었다. </li><li> MetaMorph 소프트웨어는 transfected 세포의 형광 이미지를 매 15 초 캡처하는 데 사용되었다. </li><li> 자외선에 의해 흥분 형광 염료로 코팅 유리 접시를 사용하여 조명 장소의 좌표와 크기를 정의합니다. 크기는 수다른 배율로 객관 렌즈를 적용하여 서로 다른 코어의 크기와 광학 섬유를 선택하여 다양한됩니다. </li><li> 세포 주변에 막 ruffling, 비추다의 자외선 빛을 배경 수준을 결정 후에. 다음 두 단계화된 효과를 이전 대조하는 것입니다. </li><li> 글로벌 수은 램프에서 제공하는 365 nm의 조명과 함께 세포를 조사하다. </li><li> , uncaged DMSO의 후 PBS (400 nm의 결승전)에 따라서 원래의 rapamycin를 해산하고, 글로벌 효과를 유발하는 이미징 챔버에 솔루션을 추가합니다. </li></ol> 4. 대표 결과 대표적인 결과의 예제는 그림 3에 표시됩니다. 셀룰러 가장자리 근처의 작은 지역에서 자외선의 조사가 급속화된 형성을 유도하는 것은 조사 지역 및 인근에만 주름. 이 whi 모두, 우리는 그때 전세계 자외선으로 세포를 반구, 실제로 현지 활동이었다, 또는 대량 매체에 rapamycin을 추가했는지 확인하려면채널은 세포막에 걸쳐 세계적인 주름 형성을 유도. 정량 분석​​을 위해, 우리는 네 quadrants로 타겟 세포를 나누어, 그리고 주름의 현지시 각 사분면에 형성뿐만 아니라 컨트롤 (그림 4) 등 다양한 조건 하에서 글로벌 자극을 다음의 주파수를 계산. 그것은 오직이 자외선 조사의 조합, cRb-, 지방 ruffling을 유도 적절한 FKBP-FRB 구조입니다. <img alt="그림 1" src="/files/ftp_upload/3794/3794fig1.jpg" /> 그림 1. 갇힌 rapamycin-아비딘 켤레축 (cRb-)과 자외선을 사용 spatially 국한 단백질 dimerization의 도식. 자외선 조사는 화학 dimerizer (HE-Rapa, C40-O-(2 - 히드록시 에틸) rapamycin)과 그 부산물의 릴리스 결과, rapamycin과 비오틴 사이의 링커를 클리브. 발표 dimerizer 다음 세포로 diffuses 및 FKBP-POI (관심있는 단백질)와 플라즈마 memb 사이에 dimerization을 유도rane은 조사 지역 (파란색 동그라미)의 근처에 FRB를 정박. UV 조사없이 FKBP와 FRB는 따라서 아무런 효과를 생산 안되고, (적십자) 연관시키지 않습니다. (저작권 @ ACS2011) 6 <img alt="그림 2" src="/files/ftp_upload/3794/3794fig2.jpg" /> 그림 2. 맞춤 공촛점 형광 현미경 사진. 공촛점 현미경 () 정면보기 (Axiovert 200, 자이스 혈구를 거꾸로 읽었). 에피 형광 및 자외선 조명을위한 커플링 단위의 (b)는 클로즈업, 최고 볼 수 있습니다. <img alt="그림 3" src="/files/ftp_upload/3794/3794fig3.jpg" /> 그림화된 자외선 조사에 의해 유도된 transfected NIH3T3 세포 3. Spatially 국한 주름 형성. YFP 태그가 추가된 FKBP-Tiam1 (RAC 활성)과 LDR (FRB를 멤브레인 - 고정)으로 transfected되는 NIH3T3 fibroblast 세포의 공촛점 형광 이미지는 L과의 가장자리 근처의 좁은 지역에서 조사를 받았습니다15 초이 대량 미디어 cRb-켤레축의 면전에서 210 초부터 시작을위한 365 nm의에서 ED 라이트. 이 치료는 1560 초 rapamycin 최종 400 NM의 1063 초 및 글로벌 또한 1 초위한 수은 램프와 글로벌 일루미 네이션 다음되었다. 세포는 처음 ()에서 거의 ruffling 활동을 보여주 없지만 보여주 spatially화된 조명 후에 ruffling 국한 (노란색 동그라미) (B). 세포는 로컬 주름 형성은 RAC의 spatially 제한된 활성화로 인한 것을 나타내는 글로벌 일루미 네이션과 rapamycin (CD)가 추가되는 이후 글로벌 주름 형성을 보여주었다. 스케일 바 : 10 μm의. (저작권 @ ACS2011) 6 <img alt="그림 4" src="/files/ftp_upload/3794/3794fig4.jpg" /> 그림 4. transfected NIH3T3 세포의 quadrants의 주름 형성의 빈도. cRb-A와 다음 글로벌 자외선 조사는 13 세포에서 수행되었다. cRb -와 로컬 자외선 조사 </s&gt; 또한 (노 자외선 조사)를 tron​​g, 아비딘 여부에 상관없이 현지 자외선 조사는 각각 13, 5, 6 세포에 구현되었다. 관찰된 세포들은 아무도 자외선 조사, 또는 함께 또는 대용량 미디어 아비딘없이 현지 자외선 조사없이 cRb-으로 주름 형성을 보여주 없습니다. 우리는 낮은 배경 주름 형성과 함께 세포에서 데이터를 수집하지만, 그건 우리가 rapamycin (최종 400 NM)의 이외의 결과로 세계적인 주름 형성을 확인하여 각 실험 후 확인 rapamycin 유발 주름 형성의 용량을했습니다. 이러한 결과는 cRb - 지방 자외선 조사가 세포의 지방 주름 형성에 필요한 것을 보여주었다. (저작권 @ ACS2011) 6 약어 : FKBP : FK506 결합 단백질 FRB : FKBP-rapamycin-결합 단백질 Tiam1 : T-세포 림프종의 침략과 전이 - 유도 단백질 1 (RAC GEF) LDR : 린 - DSAG 링커-FRB (린 : 린 키나제의 N-터미널 11 아미노산) HE-Rapa : C40-O - (2 - 히드록시 에틸) rapamycin cRb : 비오틴 잔기와 갇힌 rapamycin cRb-: 아비딘하기 cRb 복합

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관심의 어떠한 충돌 선언 없습니다.

우리는 초 시간 척도에 대한 정밀한 subcellular 위치에 Rho GTPase 활동을 조작하기 위해 FKBP-FRB heterodimerization 시스템 소설 갇힌 화합물을 함께 고용 기법을 설명했다. 현재의 접근 방법의 세 한계가 있습니다. 메서드가 플라즈마 막 걸쳐 dimerizer의 확산을 방지하거나 허용을 기반으로하고 있기 때문에 첫째, 대상 세포 위치는 플라즈마 막 또는 그 주변 있어야합니다. cRb의 화학 구?...

<table border="1"><tbody><tr><td> 시약 / 장비의 명칭 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Rapamycin </td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE HD </td><td> 로슈 </td><td> 04709691001 </td></tr><tr><td> 아비딘 </td><td> 시그마 </td><td> A9275-10MG </td></tr><tr><td> DMSO </td><td> 시그마 </td><td> D2650-5X5ML </td></tr><tr><td> 크기 - 배제 칼럼 </td><td> GE 헬스케어 </td><td> 스핀 트랩 G-25 </td></tr><tr><td> 유리 바닥 8 아니라 챔버 </td><td> 써모 과학 </td><td> 12-565-47 </td></tr><tr><td> 간접 형광 현미경 </td><td> 자이스 혈구 </td><td> Axiovert200M </td></tr><tr><td> 자외선은 광원을 LED가 </td><td> Rapp Opto 전자 </td><td> UVILED </td></tr><tr><td> 공촛점 스피닝 디스크 </td><td> Yokogawa 전기 주식 회사 </td><td> CSU10 </td></tr><tr><td> CCD 카메라 </td><td> 하마 마츠 Photonics </td><td> 오카-ER </td></tr><tr><td> 100x 목적 렌즈 </td><td> 자이스 혈구 </td><td> 계획 고차 색지움 </td></tr></tbody></table> cRb의 합성 구조 (저작권 ACS2011) 6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="계획 1" /></a> 합성 조건 : () BnBr, K 2 CO 3, DMF (B) f.HNO 3 AcOH (C) TFA (D) Propargyl-BR, K 2 CO 3, DMF, (마) 글리세롤, 고양이.에게 pTsA, 톨루엔 (F) NaBH 3 CN, TiCl 4, MeCN (G) TF 2 O, Pyridin (H) 메탄올 HCL,은 (i) LiAlH 4, THF (J) TsCl, 피리딘, 채널이 망할 CIA 2 (K) 앤 3, DMF (L) 8, 2,6 - 디-T-butylpyridine, 채널이 망할 CIA 2 (M) 13 CuSO 4, 아스코 브 산, 2 - 프로파놀, H 2 O, CH 2 CIA 같으니 2.

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

위대한 spatiotemporal 정밀도와 작은 구아 노신의 triphosphatases (GTPases)의 Rho 가족의 동적 규제는 다양한 세포 기능과 이벤트가 1, 2 필수적입니다. 그들의 spatiotemporally 동적 특성은 실시간 3 그들의 활동 및 현지화의 시각화에 의해 발표되었습니다. 분자 수준에서 다양한 세포 기능에서의 역할 깊은 이해를 얻을하려면, 다음 단계는 정확한 subcellular 위치와 타이밍에 단백질 활동의 섭동 있어야합니다. (1) rapamycin-유도 FKBP-FRB의 heterodimerization와 rapamycin (2) 사진 caging 방법 : 이러한 목표를 달성하기 위해, 우리는 빛을 유발, 두 기술을 결합하여 작은 GTPases의 spatio-temporally 제어 활성화를위한 방법을 개발했습니다. rapamycin-매개 FKBP-FRB의 heterodimerization의 사용으로, 우리는 급속하게 작은 GTPases includi의 활성화 또는 불활 성화를 inducible하는 방법을 개발했습니다NG RAC 4, rapamycin은 FRB가 정박되어 플라즈마 막까지, FKBP-융합된 GTPases, 또는 그들의 activators의 translocation을 유도하는 Cdc42 4, RhoA 4 RAS 5. 이 heterodimerization 시스템과 커플링 들어, 우리는 또한 rapamycin의 analogs의 사진 caging 시스템을 개발했습니다. 사진 갇힌 화합물은 누구 활동 caging 그룹으로 알려진 photocleavable 보호 그룹과 억압되어 작은 분자이다. 완전히 heterodimerization 활동을 억제하기 위해, 우리는 세포 6 안의 화학 dimerizer로 거의없이 백그라운드 작업으로 이어지는, 그 결과 대규모 복합 단지 플라즈마 막 건너없는 그런 macromolecule에 닿는 곳에있다 갇힌 rapamycin을 설계했습니다. 그림 1은 우리의 방식을 보여줍니다 시스템. 이 두 시스템의 결합을 통해, 우리는 로컬 초 timescale에 플라즈마 막에 RAC 활성을 모집하고 subcellular 레바에 빛을 유발 RAC 인증을 획득엘 6.

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

빛으로 작은 GTPase 활동 spatio-시간적 제어를위한 방법을 설명합니다. 이 방법은 rapamycin-유도 FKBP-FRB의 heterodimerization 및 사진 caging 시스템을 기반으로합니다. 빛을 조사의 최적화는 subcellular 수준에서 작은 GTPases의 spatio-temporally 제어 활성화를 가능하게합니다.

리빙 셀의 Subcellular 수준과 초 Timescale에 작은 GTPase 활동의 Spatio-시간적 조작

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

spatio-temporal-manipulation-small-gtpase-activity-at-subcellular

Research

JoVE Journal

Bioengineering

10.7K 조회수.  Johns Hopkins University. 빛으로 작은 GTPase 활동 spatio-시간적 제어를위한 방법을 설명합니다. 이 방법은 rapamycin-유도 FKBP-FRB의 heterodimerization 및 사진 caging 시스템을 기반으로합니다. 빛을 조사의 최적화는 subcellular 수준에서 작은 GTPases의 spatio-temporally 제어 활성화를 가능하게합니다.

비디오: Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

생활 세포의 분자 Rotors의 형광 수명 이미징

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

연구

생체공학

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

원자 힘 현미경을 사용하여 살아있는 세포의 기계적 특성을 측정

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

단일 베이 수준에서 산소 미시의 양 및 시간 제어

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

생활 효모 세포에서 미토콘드리아 산화 환원 상태와 ATP를 측정 비례 바이오 센서

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

수술 조직에서 기본 인간 대장 종양 세포의 분리 및 견인 세포 계측법 부드러운 탄성 기판에 직접 배양

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

단일 세포 분비물의 공간 - 시간 영상의 레이블이없는 기술

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

공액 고분자에 의해 세포를 전기적 활동을 생활의 광 제어

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

에 Microengineered 인간의 장내 융모와 생활 마이크로 바이 옴의 공동 문화 창자 - 온 - 어 - 칩 미세 유체 장치

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

인간의 치경과 파생 셀 Osteogenic 활동에 비타민 D의 효과에서 중간 엽 줄기 세포의 분리

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

살아있는 세포 형광 기술의 조합을 사용 하 여 힘에 민감한 단백질 역학의 측정

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...