This work was supported by NIH HL-10342

참고 : 모든 동물 프로토콜은 존스 홉킨스 대학 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. 동물 준비 <ol><li> 아래의 단계 2.3에 설명 된대로 케타민과 자일 라진으로 그들을 마취에 의해, DFCO 측정을 위해 6 C57BL / 6 컨트롤 마우스를 준비합니다. </li><li> 컨트롤과 동일한 방법을 사용하여 표 1에 나타내는 다른 폐 병변을 가진 다른 모든 실험군을 준비한다. 이들 각각의 모델을 구축하기 위해 필요한 특정 세부 사항이 관련 참고 문헌에서 발견된다. 제어 마우스 및 기타 병리학 적 코호트에서 그 나이의 모든 6~12주 있습니다. </li></ol> 일산화탄소에 대한 확산 계수 2. 측정 (DFCO) <ol><li> 질소, 산소, 네온, 및 일산화탄소에 대한 피크를 측정하는 기기에 공급되는 가스 크로마토 모듈을 설정한다. 이 응용 프로그램에만 사용 네온 및 CO 데이터. 참고 :이 장비는 분자의 사용12.00 μm의 필름, 320.00 μm의 ID와 10m 길이의 캐리어 가스로 헬륨 ieve 열. 크로마토 그래피 컬럼은 0.8 ml의 부피를 가지고 있지만, 우리는 샘플 튜브의 적절한 연결을 보장하기 위해 클리어 2ml를 사용했다. </li><li> 각 실험 하루의 시작시 마우스의 샘플의 측정을하기에 앞서, 약 0.5 %의 Ne, 0.5 % CO를 함유하는 가스 혼합물 백에서 직접 2 ㎖의 샘플을 채취하고, 공기를 밸런스, 보정이 샘플을 사용 가스 크로마토 그래피. </li><li> 케타민 (90 ㎎ / ㎏)과 자일 라진 (15 ㎎ / ㎏)와 쥐를 마취하고, 반사 운동의 부재로 마취를 확인합니다. 건조 함을 방지하기 위해 눈에 수의학 연고를 적용합니다. (아주 어린 쥐에서 성인의 18 G, 20 G) 스텁 바늘 캐 뉼러와 마우스를 Tracheostomize. 참고 : DFCO 마취 후 미만 10 분에 완료하고 기계 환기, 또는 다른 절차에 앞서있다. </li><li> 6 주령 마우스에서보다 크고, 3 ㎖ 주사기를 사용하여 tO 혼합 가스 주머니로부터 기체를 인출 2,3,4-. 기관 정맥에 주사기를 연결하고 신속하게 폐를 팽창. 메트로놈을 사용하여, 9 초를 계산 한 다음 신속하게 0.8 ㎖ (호기, 공기)을 철회. </li><li> 이 철회 0.8 ml의 실내 공기와 2 ml의 공기를 내뿜어 희석, 적어도 15 초 동안 휴식 할 수 있습니다. 그 후 분석을 위해 가스 크로마토 그래피로 시료를 주입 전체. </li><li> 이 제 DFCO 샘플을 분석하는 동안, 가스 혼합물로부터 제 가방 0.8 ml의 마우스 폐 팽창하고 첫 번째 샘플이 동일한 샘플을 처리한다. 두 DFCO 측정에 대한 평균값. 주 : 0.8 ml의 부피가 매우 어린 마우스의 폐에서 측정을 수행하기에 너무 크기 때문에 생후 이주로 어린 마우스에서의 측정을 위해, 0.4 ml의 부피를 사용한다. 그것은 6주보다 오래된 쥐 0.8 ㎖의 부피를 사용하는 것이 더이며, 0.4 ml의 부피가 약간 생쥐에 필요하면,이 연구되고 코호트 모든 생쥐 일관 사용해야 함. </li><li> DFCO를 계산(CO 9 / CO의 C) / (느 9 / 네브라스카 C), C와 9 첨자가 주입 된 교정 가스의 농도가 각각 9 초 숨 유지 시간 후에 제거 가스를 참조 - 1 등. </li><li> 분석하고 일방향 ANOVA와 차이점을 비교하고 모든 코호트 마우스에서 다중 비교에 대한 보정 Tukey의 유의 수준을 평가. P &lt;0.05과 같은 중요한 가치를 고려한다. 참고 : 여기에 사용 된 마우스의 모든 여기에보고되지 않은 폐 환기, 기계, 폐 세척, 또는 조직 학적 여러 후속 측정을 포함하는 실험 연구의 일부. 상기 방법은 대조군 마우스 위에서 행해졌 모든 실험 모델에서 동일하기 때문에, 이러한 다양한 병리 모델 만 결과가 제시된다. 이 모델에 대한 추가 정보는 보조 표에 표시됩니다. </li><li> 깊은 anesthe에 의해 동물을 안락사TIC 과다 복용은 자궁 경부 전위 또는 잘린 하였다. 필요한 경우, 추가 생물학적 또는 조직 학적 처리 및 분석을 위해 죽은 쥐에서 폐 세포 및 / 또는 조직을 제거합니다. </li></ol>

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관심 아무것도 없음 충돌은 공개가 없습니다.

본 연구에서는 마우스 폐의 가스 교환 능력을 정량화하는 새로운 메트릭을 정의했다. 이 메트릭은 확산 능, 즉 폐의 주 기능을 측정하는 일반적인 임상 측정 가스를 교환 할 수있는 능력과 유사하다. 확산 능 쉽고 빠르게 마우스 및 인간 모두에서 수행 될 수있는 유일한 폐 기능의 측정이다. 마우스의 폐 질환을 검출하기위한, 주요 목적은 제어 및 실험 코호트 간의 폐 기능의 변화를 정량화하는 ?...

이제 마우스 폐 질환의 다양한 모델링하는데 사용되는 기본 동물이다. 이러한 병리를 underly 메커니즘을 연구하기 위해, 표현형 방법은 그것을 병리학 적 변화를 정량화 할 수 필요합니다. 환기 역학을 측정 마우스 많은 연구가 있지만, 이러한 측정들은 일반적으로 인간 정상적으로 이루어 폐 기능의 평가 표준과 무관하다. 이것은 인간의 질병에 마우스 모델에서의 번역 결과를 용이하게 할 수있다 마우스 및 인간 대상에서 동등한 측정을 수행 할 수있는 능력 때문에, 유감. 피험자의 가장 일반적이고 용이하게 측정 중 하나는 일산화탄소 (DLCO) 1,2 확산 능이지만,이 측정은 거의 마우스 모델에서 수행되지 않았다. 절차는 종종 복잡하거나 REQ 수 있기는 3-7보고되었다 이러한 연구에서, 부분적으로, 어떠한 후속 연구는 없었다복잡한 장비를 uire. 또 다른 방법은 민감한 마우스에서 CO 확산을 측정 할 수 있다는 장점을 갖는다 정상 상태 시스템에서 CO 재 호흡 방법을 사용하는 것이다. 그러나이 방법은 매우 복잡하고, 결과는 마우스의 환기 수준뿐만 아니라 O 2, CO 2 농도 8,9에 따라 달라질 수 있습니다. 이러한 어려움은 여러 가지 장점에도 불구하고, 마우스의 폐 병변을 감지하는 능력을 확산의 일상적인 사용을 배제 것 같다. 마우스에서의 용량의 측정과 확산 문제를 피하기 위해, 간단한 방법의 세부 사항을 마우스에서 측정하기 위해 최근 10보고되었다. 절차는 신속히 전체 영감 가스 동일한 부피를 샘플링함으로써 오염 폐포 가스 채취 어려운 문제를 제거한다. 매우 재현성있는 측정이 프로 시저의 결과, PA의 호스트에 민감 일산화탄소 (DFCO)에 대한 확산 인자로 지칭폐 표현형 thologic 변경됩니다. (CO 9 / CO의 C) / (느 9 / 네브라스카 C), C 및 9 첨자가 주입 된 교정 가스의 농도와 9 초 숨 유지 시간 후에 제거 가스를 참조 - DFCO 따라서 1과 같이 계산된다 각각. DFCO 1은 모든 CO의 완전한 흡수를 반영하고, 0은 어떠한 CO의 흡수를 반영하지으로 0과 1 사이에서 변화 무 차원 변수이다. 이 프레젠테이션에서 우리는이 확산 능 측정을하는 방법을 보여줍니다, 그것은 거의 모든 기종, 섬유증, 급성 폐 손상 및 바이러스 및 곰팡이 감염을 포함하여 기존의 마우스 폐 질환 모델의 변화를 문서화하는 방법을 사용할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gas Chromatograph</td>
			<td>Inficon</td>
			<td>Micro GC Model 3000A</td>
			<td>Agilent makes a comparable model</td>
		</tr>
		<tr>
			<td>18 g Luer stub needle</td>
			<td>Becton Dickenson</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
		<tr>
			<td>3 mL plastic syringe</td>
			<td>Becton Dickenson</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
		<tr>
			<td>Polypropylene gas sample bags</td>
			<td>SKC</td>
			<td>1 or 2 liter capacity works well</td>
			<td>Other gas tight bags will work well</td>
		</tr>
		<tr>
			<td>Gas tank, 0.3% Ne,0.3% CO, balance air; (size ME)</td>
			<td>Airgas, Inc</td>
			<td>Z04 NI785ME3012</td>
			<td>This is the standard mixture used for DLCO in humans</td>
		</tr>
		<tr>
			<td>25 TCID50/mouse of influenza virus A/PR8 diluted in phosphate buffered saline.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine pancreatic elastase</td>
			<td>Elastin Products, Owensville, MO</td>
			<td></td>
			<td>5.4 U</td>
		</tr>
		<tr>
			<td>Bleomycin</td>
			<td>APP Pharmaceuticals, Schaumburg, IL</td>
			<td></td>
			<td>0.25 U</td>
		</tr>
		<tr>
			<td>Escherichia coli LPS8</td>
			<td>Sigma L2880</td>
			<td></td>
			<td>3 &#956;g/g body weight; O55:B5</td>
		</tr>
		<tr>
			<td>Aspergillus fumigatus (isolate Af293) conidia were collected from mature colonies grown on potato dextrose agar.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

phenotyping mouse pulmonary function in vivo with the lung diffusing capacity

이제 마우스 폐 질환의 다양한 모델링하는데 사용되는 기본 동물이다. 이러한 병리의 기초 메커니즘을 연구하기 위해, 표현형 방법은 병리학 적 변화를 정량화 할 수 필요합니다. 게다가, 마우스 모델 병진 관련성을 제공하기 위해, 이러한 측정은 쉽게 인간 및 마우스 모두에서 시험을 수행 할 수 있어야한다. 불행하게도, 본 문헌에서 폐 기능의 몇 가지 표현형 측정은 인체에 직접 응용 프로그램이 있습니다. 한 가지 예외는 일상적으로 인간에서 수행되는 측정 일산화탄소에 대한 확산 용량입니다. 본 보고서에서는 신속하고 간단하게 마우스에서이 확산 능력을 측정하는 방법을 설명한다. 절차는 1 분 가스 분석 시간 뒤에 마취 마우스의 추적 가스와 간단한 폐 인플레이션을 포함한다. 우리는 폐기종, 섬유증, 급성 폐 손상, 인플루엔자 등 여러 가지 폐 병변을 감지하는이 방법의 능력을 테스트 한진균 폐 감염뿐만 아니라, 젊은 새끼에서 모니터링 폐 성숙. 결과는 상당한 모든 폐 병변의 감소뿐만 아니라 폐 성숙 확산 용량 증가를 나타낸다. 폐 확산 능의이 측정 따라서 기존의 병리학 적 폐 모형의 대부분과 표현형의 구조적인 변화를 감지 할 수있는 능력과 폭 넓은 응용 프로그램이있는 폐 기능 검사를 제공합니다.

도 1이 퍼질러 인플루엔자 감염 모두 상당한 감소뿐만 아니라 섬유증, 폐기종에서 상당한 감소가 있었고, 급성 그룹 A, B, C, D, E, 및 F.에서 성숙한 생쥐 DFCO 측정치를 나타낸다 폐 손상 모델. 그림 2는 2~6주 쥐 나이 시간에 DFCO의 그룹 G 발달 변화를 보여줍니다. 이 때 코스 위에 폐 개발 약간이지만 상당한 증가가 있었다. 작은 팽창 체적의 사용 효과는 6 주 시점에서 아주 명?...

Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

표현형 마우스 폐 기능<em&gt; 생체</em&gt; 폐 확산 능력

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

phenotyping-mouse-pulmonary-function-vivo-with-lung-diffusing

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JoVE Journal

Biology

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

10.5K 조회수.  Johns Hopkins University Bloomberg School of Public Health. We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

비디오: Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

연구

생물학

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

절연, 환기 및기구 마우스 폐의 특성화는 타악기 흐름 Perfused

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

대형 Unilamellar 소포와 검사 BCL-2 가족 기능

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

생물 전자 현미경에서 동결 골절 / 동결 에칭의 기본 기술 요소

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아의 분리

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

은 C-FOS 단백질 면역 조직 검색 : 특정 생리적 반응에 관여 중앙 진학의 마커로서 유용한 도구<em&gt; 생체</em&gt; 및<em&gt; 전의 VIVO</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

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