우리는 이러한 연구들의 관대한 지원 ReadiSorb 제품 감사합니다.

1. 세포 고양이 astrocytes의 문화와 H 2 O 2와 치료 <ol><li> 37 DMEM 미디어 플러스 영양소와 75cm이 플라스크에 문화 세포주 G355 - 5 ° C와 5% CO 2 60 % 합류 (~ 5 일)까지. 모든 1~2일 미디어를 교체 또는 건강한 문화를 유지하는 데 필요한. </li><li> 미디어를 제거하고 0.25 %의 트립신 2 ML를 추가합니다. 45 S에 대한 pipetting하여 세포를 리프트 - 실내 온도 (RT)에서 1 분. </li><li> 신속 미디어 10 ML을 포함한 15 ML 원뿔 관에 현탁액을 전송하기만하면됩니다. 부드럽게 섞는다. </li><li> 펠렛을 얻기 위해 3 분 300 XG, 23 ° C에서 원심 분리기. 펠렛을 방해하지 않고 뜨는 폐기하십시오. </li><li> 신선한 미디어 6-7 ML을 추가하고 세포를 resuspend. </li><li> 6 잘 플레이트의 각 잘하는 셀 ~ 1 ML을 추가합니다. 각 물론 미디어로부터 2-3 ML을 추가하고 80~90%의 합류까지 품어. </li><li> 미디어 과산화수소의 100μM 솔루션 (H 2 O 2)를 준비합니다. </li><li> 각 우물에서 미디어를 제거하고 하나 둘 ML 신선한 매체 (제어), H 2 O 2 (치료) 또는 DPBS의 2ml 2 ML로 바꿉니다. ° C, 5 % CO 2 3 H. 위해 37 알을 품다 </li><li> 솔루션을 제거하고 세포를 리프트하는 트립신 1 ML를 추가합니다. 0.5 ML의 DPBS을 포함하는 1.7 ML Eppendorf 튜브에 세포를 전송합니다. 이 단계는 연장 트립신 보육에서 세포 죽음을 방지하기 위해 각 잘 개별적에서 수행되어야합니다. </li><li> 3 분 300 XG에 스핀, 버리고 뜨는 및 DPBS 1 ML에 resuspend. </li></ol> 2. ROS를위한 염색법 <ol><li> 얼룩의 모든 단계 표백 방지하기 위해 최소한의 빛의 노출을 실시해야합니다. </li><li> 긍정적인 제어 50 MM의 CCCP 1 μl를 추가합니다. 37 품어 ° C, 5 % 5 분 CO 2. </li><li> H 2 O 2 그룹 10 MM CM - H 2 DCFDA 솔루션 5 μl를 추가합니다. 37 품어 ° C 5 % CO 15-30 분 2. 아무 얼룩은 생리 예제에 추가하지 않습니다. </li><li> 잔여 얼룩을 제거하는 DPBS 2 ML과 샘플을 씻으십시오. </li><li> 펠렛을 얻기 위해 3 분 300 XG에 스핀. </li><li> DPBS 0.5 ML에서 세포를 Resuspend. 생리를 제외하고, 각 예제 1 μl PI를 추가합니다. </li><li> 5ml 흐름 cytometer 튜브에 샘플을 전송합니다. </li><li> 흐름 cytometer를 실행합니다. </li></ol> 3. 유동세포계측법 <ol><li> 525, 775 및 620 nm의 모든 ± 20 nm의 : 488 nm의 아르곤 레이저 다음과 같은 밴드 패스 갖춘 베크맨 쿨터 FC500 (또는 이에 상응하는 금액)에서 샘플을 실행합니다. </li><li> 해당 컨트롤을 준비 : 흠없는 세포가, CM - H 2 DCFDA은 (대조군) PI 단, 더블 스테인드 밖에 스테인드. </li><li> FS 대 SS, PI 대 CM - H 2 DCFDA 다음 histoplots을 가진 프로토콜을 확인합니다. 또한 다음과 같은 histograms을 가집니다 얼룩에 해당하는 휴대폰 번호 대 FL1, FL2 및 FL3을. </li><li> 샘플을 실행하기 전에, 장비, 유체 및 폐기물 컨테이너를 확인합니다. 최소한 20 분 장비를 따뜻하게. </li><li> 장비를 청소하고 제조 업체의 표준에 따라 적절한 주판으로 보정을 수행합니다. </li><li> 귀하의 프로토콜을 선택하고 샘플을 실행합니다. </li><li> 이전 완료로 장비를 청소하십시오. </li><li> 제조 업체의 기준에 따라 진공 라인을 청소하십시오. </li><li> 흐름을 끄고 머리 / 진공 라인을 청소하십시오. </li><li> 소프트웨어 및 컴퓨터를 끕니다. </li></ol> 4. 대표 결과 : 유동세포계측법 결과는 FlowJo 7.6과 얼룩 컨트롤이 객관적으로 게이팅을 설정하는 데 사용되었다를 사용하여 분석했다. 이것을 근거로, 건강한 세포가 히스토그램 및 ROS (산화 스트레스)의 왼쪽에 표시가 오른쪽으로 세포의 변화로 감지되었다. 그림 1은 건강한 고양이 astrocytes에 과산화수소의 효과의 결과를 보여줍니다. FL1에서 데이터는 ROS의 존재를 나타내는 CM - H 2 DCFDA의 강도를 측정하는 데 사용되었다. 예상했던대로, ROS의 높은 금액은 H 2 O 2 대우 샘플에서 발견되었다. 이것은 왼쪽에서 오른쪽으로 형광 강도의 변화로 히스토그램에 표시됩니다. DMEM 대 DPBS 취급 건강한 세포를 비교하면, ROS의 양을 (그림 2)에는 큰 변화가 없었다. <img src="/files/ftp_upload/2841/2841fig1.jpg" alt="그림 1"/> 건강한 고양이 astrocytes에서 산화 스트레스의 수준과 건강한 세포에 H 2 O 2의 효과 그림 1. 유동세포계측법 결과. 결과없이 치료 (DMEM)과 세포와 비교하여 H 2 O 2 건강한 세포에 ROS의 수준을 증가시킬 수있다는 것을 나타냅니다. <img src="/files/ftp_upload/2841/2841fig2.jpg" alt="그림 2"/> DPBS와 3hrs에 대한 incubated 건강한 세포에 감지 ROS의 수준의 그림 2. 유동세포계측법 결과.

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바이러스 자주 제안된 메커니즘의 연결 손상이 산화 스트레스 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31 유도. 기본적으로, 그것은 제안 이러한 수산화, 초과 산화물의 음이온, 질산 산화물 및 / 또는 신경에​​ 독성이 과산화수소, 같은 바이러스성 노출을 통해 glia (astrocytes와 microglia)가 릴리스 반응 산소 래디 칼. 마찬가지로, 산화 스트레스는 또한 암페타민 유발 neurotoxicity 2, 6, 17의 주요 patho...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013 - CV </td><td></td></tr><tr><td> 멤 비타민 </td><td> Cellgro </td><td> 25-020 - CL </td><td> 1퍼센트 (V / V) </td></tr><tr><td> L - 글루타민 </td><td> Hyclone </td><td> 17 - 605E </td><td> 1퍼센트 (V / V) </td></tr><tr><td> 비 필수 아미노산 </td><td> Hyclone </td><td> 25-025 - CL </td><td> 1퍼센트 (V / V) </td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td> 20% (V / V) </td></tr><tr><td> 필수 아미노산 </td><td> Cellgro </td><td> 25-030 - CL </td><td> 0.2 % (V / V) </td></tr><tr><td> 500ml 진공 여과 시스템 </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ML 원뿔 튜브 </td><td> 매 </td><td> 352097 </td><td></td></tr><tr><td> 75cm 두 조직 문화 flasks </td><td> 매 </td><td> 353136 </td><td></td></tr><tr><td> 6 잘 조직 배양 플레이트 </td><td> 매 </td><td> 353224 </td><td></td></tr><tr><td> CM - H 2 DCFDA </td><td> Invitrogen </td><td> C6827 </td><td></td></tr><tr><td> Propidium의 이우 (PI) </td><td> 시그마 </td><td> P4170 - 10mg </td><td></td></tr><tr><td> 트립신 </td><td> Lonza </td><td> 17 - 160F </td><td></td></tr><tr><td> H 2 O 2 </td><td> 시그마 </td><td> H6520 </td><td></td></tr><tr><td> HyQ의 항생제 </td><td> Hyclone </td><td> SV30079.01 </td><td> 0.1 % (V / V) </td></tr><tr><td> G355 - 5 전지 </td><td> ATCC </td><td> CRL - 2033 </td><td> 일반 고양이 뇌 </td></tr></tbody></table>

screening assay for oxidative stress in a feline astrocyte cell line, g355-5

의 연결을 손상을 유도 바이러스의 자주 제안 메커니즘은 산화 스트레스입니다. Astrocytes는 중추 신경계 (CNS)의 산화 스트레스를 조절에 중요​​한 역할을했습니다. Astrocytes는 반응 산소 종 (ROS)에서 뉴런뿐만 아니라 보호하는 뉴런에 생체 항상성 환경을 유지할 수 있도록 도와주십시오. CM - H2DCFDA는 ROS의 존재에 대한 세포 침투성 표시기입니다. CM - H 2 DCFDA이 아닌 형광 화합물로 세포를 입력하고, 휴대폰 esterases은 아세테이트 그룹을 제거한 후 형광되고, 화합물이 산화됩니다. 녹색 fluorescing 것으로 판단될 유동세포계측법에 의해 측정한 세포의 숫자는 산화 상태에있는 세포의 수를 나타냅니다. CM - H 2 DCFDA 다른 ROS 다수에 의해 산화 쉽습니다. 이 분석에 관계없이하는 산소의 산화 세포 환경을 화면으로 사용할 수로 ROS는 CM - H 2 DCFDA을 산화 수있는에 대한 특이성의이 부족은,이 화합물 pathogenesis 조사의 초기 단계에 사용하기 위해 가치있는 리젠트합니다 ROS의 급진 또는 조합은 세포 조건에 책임이 있습니다. 일단 그것은 ROS가 CM - H 2 DCFDA의 산화에 의해 현재 다음 추가 실험이 ROS 또는 로스의 조합이 특정 pathogenesis 과정에 참여하는 결정하기 위해 수행할 수있는 것으로 설립되었습니다. 본 연구의 결과는 과산화수소 CM - H 2 DCFDA의 증가 또한이 분석은 G355 - 5 세포 내에서 산화 환경을 검출을위한 가치있는 테스트입니다 나타내는 생리 컨트롤에 상대적으로 감지되었습니다 형광 것을 입증 고양이 astrocyte 세포 라인.

Watch this Scientific Journal Video about Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5 at JoVE.com

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

산화 세포 환경을 감지하는 심사 방법은 CM - H2DCFDA의 산화를 측정하는 것입니다. 일단 비 형광의 형광 화합물로 세포, CM - H2DCFDA 변경 내에 산화. 형광에이 변경 사항은 유동세포계측법으로 측정하고 산화 환경에서 세포의 수를 나타냅니다.

고양이 Astrocyte 세포주에서 산화 스트레스에 대한 심사 분석, G355 - 5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

screening-assay-for-oxidative-stress-feline-astrocyte-cell-line-g355

Research

JoVE Journal

Neuroscience

14.3K 조회수.  Western University of Health Sciences. 산화 세포 환경을 감지하는 심사 방법은 CM - H2DCFDA의 산화를 측정하는 것입니다. 일단 비 형광의 형광 화합물로 세포, CM - H2DCFDA 변경 내에 산화. 형광에이 변경 사항은 유동세포계측법으로 측정하고 산화 환경에서 세포의 수를 나타냅니다.

비디오: Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast 분석 : 유동세포계측법을 사용하여 차별화 신경 줄기 세포에서 자손의 연결 전구 세포의 생성과 강화에 대한 분석

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

연구

신경과학

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

쥐과 장내 신경 전구 세포에 대한 양이 세포 이동의 분석

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

신경 세포주에 신경 독성과 신경에 관여하는 분자 진학의 윤곽을하는 도구로 실시간 임피던스 기반의 세포 분석기

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

간접 신경-성상 세포 공동 배양 분석 일 :<em&gt; 체외</em&gt; 신경 세포 - glia 상호 작용의 상세한 조사를위한 셋업

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

기존의 UV 레이저 공 초점 현미경을 사용하여 트리거 세포 스트레스와 죽음

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

살아있는 세포에 Sod1 돌연변이 단백질 집계 형성의 높은 콘텐츠 정량화에 대 한 분석 결과 개발

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

소설 체 외에 라이브 이미징 분석 결과의 사이토 중재 먹어서 사용 하 여 pH 지시자 활용 Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

생체 외에서 타우 단백질 및 마약 검사의 집계를 공부에 대 한 분석 결과

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

애벌레 Zebrafish의 측면 라인 세포의 생체 내 칼슘 이미징

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

고양이과 거대 동물 모델에서 인간 배아 줄기 세포 유래 망막 조직의 망막하 이식

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...