입양 DTH의 유도 조브 문서를 참조하십시오 : <a href="/index/Details.stp?ID=325">고양이의 입양 지연 유형 과민성의 유도 및 모니터링</a> 크리스틴 비튼, 조지 K. Chandy 생리학 및 생물 물리학의학과 캘리포니아 대학, 어바인 J. 시각 실험, 제 8 여기, 우리는 phagocytic 항원 제시 세포의 시각화 수 있도록 레이블이 지정되지 않은 항원과 1:1 비율 항원 (Ovalbumin) 텍사스 - 레드에 복합을 사용하여이 프로토콜을 수정했습니다. 이어 조직 하베스트 <ol><li> 귀하의 승인된 동물 프로토콜에 명시된 절차에 의해 원하는 시간 지점에서 쥐를 안락사. 여기, 우리는 isoflurane 마취의 과용을 사용합니다. 이것은 통풍이 잘 후드 안에있는 닫힌 용기에 이루어집니다. 동물의 흉강을 열고 심장을 절단하여 중 잘린에 의해 죽었다는 보장합니다. </li><li> 큰 가위를 사용하면 가능한 한 동물의 몸에 가까운 싱글 컷 (그림 1)과 귀를 제거합니다. <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> 그림 1. 쥐 (内耳)의 제거 </li><li> 얼음처럼 차가운 1X PBS 또는 RPMI - 1640의 ~ 10 ML을 포함한 15 ML 원뿔 관에 귀를를 놓습니다. 이 영상에 대한 조직을 준비하는 준비가 될 때까지 얼음 조직 샘플을 보관. </li><li> 최대한 빨리 이미지를 조직, 우리는 수확 즉시 군데 조직에 비해 얼음 1로 2 시간 조직에있는 세포 운동성에 뚜렷한 효과가 없다고하지만, 찾으십시오. </li></ol> 이미징을위한 조직 준비 표피와 피부 레이어의 I. 제거 * 참고 : 깊은 피부 레이어는 그대로 보관해야합니다. 제대로하면 큰 혈관은 그대로 유지되며 귀의 연골이 노출되지 않습니다. 더 염증 (관리 규정)이없는 특히 이것은 어려운 기술이며 그것은 해부 현미경을 사용하여 도움이됩니다. <ol><li> 10cm 조직 문화 요리에있는 원뿔 관의 조직과 언론 모두를 놓습니다. </li><li> 장갑 낀 손 (그림 2) 최대 직면하고있는 귀의 지느러미 측면과 귀 (말초)의 끝에 부드럽게 쥐의 귀를하라. <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> 그림 2. 귀에에서 트림 모피 </li><li> 귀 상단, 깊은 진피에서 진피를 분리 폐쇄 더몬트 끝 # 5 가위를 사용하는 동안 지속적인 조직에 엄지손가락으로. 귀 가장자리 피부 / 깊은 진피의 오버행이있다면 또는, 이것은 집게로 파악 할 수 있으며 부드럽게 아래 조직 (그림 3)에서 이것을 분리 뽑아. <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> 그림 3. 상단 진피의 제거 </li><li> 일단 피부의 작은 지역 또는 부드럽게 제거하고 피부 dislodged에서 껍질로 피부 레이어와 집게 사이자를 가위를 사용하여 분리 단계를 반복, 기본 조직에서 분리되었습니다. </li><li> 5 × 5 mm 또는 항원 주입의 초기 사이트 (그림 4)에서 최소 3mm는 귀 조직의 큰 영역을 쉽게받을 수 있습니다. <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> 그림 4. 이미지에 대한 노출 조직 </li></ol> II. 이미징 스테이지에 조직을 확보 <ol><li> 준비 조직보다 약간 큰 크기로 플라스틱 커버 슬립 컷. </li><li> 3M Vetbond 슬라이드에 걸쳐 ™ 조직 안전한 접착제의 얇은 레이어를 확산. </li><li> 준비된 조직 (이 이미지에 가능성이있는 곳으로 가는게)의 코너를 파악, 미디어에서 제거하고, 소량은 렌즈 종이나 Kym에있는 조직의 아래쪽 초과 미디어를 제거하는 닦으십시오. </li><li> 접착제 커버 슬라이드 (즉시 서부 유럽 표준시 조직에 Vetbond 치료)로 조직의 가장자리를 터치. 부드럽게 (그림 5) 슬라이드를 만진되는 반대 엔드와, 평면 조직을 스트레칭 <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> 그림 5. 쪽지를 커버하기 위해 조직을 마운트 </li><li> 로 반전 조직 샘플 / 슬라이드 및 조직 및 슬라이드가 45도 각도로 얼굴 아래로 약하는 등 미디어 저수지에서 수영. Vetbond ™ 치료 각도에 거꾸로 조직을 개최하면 노출된 조직 표면에 경화에서 초과 접착제를 방지하는 데 도움이됩니다. 조직의 표면에 접착제가 레이저 (그림 6)을 차단합니다. <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> 그림 6. vetbond 조직 GLU 치료미디어 마른하여 전자 참고 : 단계 2-5은 마스터에 약간의 연습이 걸릴 수 있습니다. 처음 시도하기 전에 삭제 또는 추가 조직 조각과 연습을보십시오. </li><li> 슬라이드의 맨 아래에 실리콘 그리스의 DAB는 작은 금액입니다. </li><li> 재관류 챔버에서 슬라이드를 놓습니다. 재관류 미디어는 37 ° C를 흐르는하고 조직을 추가하기 전에 산소 있는지 확인합니다. 부드럽게 재관류 챔버의 바닥 및 플라스틱 커버 슬립 사이에 실리콘 그리스와 도장을 만들어 슬라이드의 가장자리 아래로 누르십시오. </li></ol> III. 두 광자 이미징 <ol><li> 밝은 - 현장 빛의 전송을 사용하여 조직의 꼭대기에 중점을두고 있습니다. 조명의 모든 전원을 끕니다. </li><li> 레이저를 켜고, 귀하의 다중 광자 현미경 시스템에 필요한 PMTs. </li><li> 높은 주문한 섬유 단백질의 두 번째 고조파 세대 (SHG) (콜라겐과 마이 오신)은 여기 레이저의 절반 파장에서 신호를 생산하고 있습니다. 우리는 현미경의 &quot;푸른&quot;채널을 사용하여 시각 수있는 450 nm의에서 SHG에 이르는, 900 nm의 여기에서를 사용합니다. 세대 SHG 것은 비선형 (2 광자) 과정이며, 따라서 형광의 2 광자 여기의 비슷한 광학 &quot;sectioning&quot;효과를 제공합니다. 병렬로 실행 원섬유이 시각되지 않습니다 반면 SHG의 절정 효율성은 원섬유는 레이저로 수직 때 발생 1. </li><li> 세포 가득한 좋은 이미지 영역을 찾아, 세포의 대부분 중앙에 있으며, 영상을 시작 귀하의 수집 영역을 설정합니다. 참고 : DTH 반응, T 세포와 다른 세포 infiltrates는 종종 다른 분야 세포 2 침투은없는하는 동안 함께 집중해야 볼 수 있습니다. 따라서 이미지에 가장 좋은 지역을 찾으려면 조직을 검색 시간이 좀 걸릴 수 있습니다. </li><li> 이미징 동안 셀 속도와 극성을 평가하여 세포 생존을위한 확인하십시오. 최소한 사진 손상을 보장하기 위해 허용되는 이미지 품질을 제공하는 낮은 레이저 파워를 사용하십시오. </li><li> 필요에 따라 새로운 조직 준비 스왑. </li></ol>

<ol>
	<li>Koo, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+the+voltage-gated+potassium+channel+Kv1.3+inhibits+immune+responses+in+vivo.">Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.</a> J Immunol. 158, 5120-5128 (1997).</li><li>Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+activation+and+T+lymphocyte+infiltration+in+allergen-induced+late+phase+skin+reactions+and+classical+delayed-type+hypersensitivity.">Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity.</a> J Immunol. 147, 816-822 (1991).</li><li>Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoaggressive+effector+T+cells+in+the+course+of+experimental+autoimmune+encephalomyelitis+visualized+in+the+light+of+two-photon+microscopy.">Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy.</a> J Neuroimmunol. 191, 86-97 (2007).</li><li>Kawakami, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+effector+cell+trafficking+and+autoantigen+recognition+within+the+unfolding+autoimmune+encephalomyelitis+lesion.">Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.</a> J Exp Med. 201, 1805-1814 (2005).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Matheu, M. P. B., A, C. G. a. r. c. i. a., Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Imaging+of+Effector/Memory+T+Cells+During+DTH+and+Suppression+by+Kv1.3+Channel+Block.">In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block.</a> Immunity. , (2008).</li></ol>

imaging effector memory t cells in the ear after induction of adoptive dth

효과기 / 메모리 T의 lymphocytes - 지연 유형 과민성 (DTH)은 주요 선수들이 CCR7하는 면역 반응이다. 여기, 우리는 쥐의 귀에 DTH 반응의 진행 상황을 유도하고 기록하는 방법을 보여줍니다. 효과기 / 메모리 T 세포 응답 - ​​이것은 CCR7의 2 광자 이미징을위한 쥐의 귀 조직의 준비 시범옵니다. 효과기 / 메모리 T 세포 라인 (비튼, C J. 시각 실험, 이슈 8) - 입양 DTH는 GFP - 레이블 OVA 특정 CCR7의 intraperitoneal 주사로 유도합니다. 전지는 다음 시각화를 허용하도록 텍사스 - 레드에 OVA와 OVA 복합의 1:1 혼합 (OVA - TR)과 호수 (제어 EAR)과 다른 하나의 귀를 주사하여 도전하기 전에 48 시간 동안 쥐의 평형을 사용할 수 있습니다 주민 항원 제시 세포. 우리는 영상 두번째 고조파 생성하여 콜라겐 섬유의 시각화와 함께 면역 반응 동안 피부 깊은 계층 내에서 세포의 운동성 유용 조직 준비 방법을 설명합니다. 귀 조직은 5 X 5mm의 사각형 (약간 큰 더이다)​​로 절단 후 37 이미징 챔버에 실리콘 그리스를 사용하여 보안과 산소 - 부풀어 오른 조직 문화 매체에 의해 superfused 아르 Vetbond ™를 사용하여 플라스틱 커버 미끄러져에 탑재되어 ° C .

Watch this Scientific Journal Video about Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH at JoVE.com

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

여기서 우리는 쥐의 귀에 지연 유형 과민성 (DTH) 반응의 진행 상황을 유도하고 기록하는 방법을 보여줍니다. 이것은 이펙터 / 메모리 T 세포 응답의 2 광자 이미징을위한 쥐의 귀 조직의 준비 시범옵니다.

입양 DTH의 유도 후 귀에 영상 효과기 메모리 T 세포

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

imaging-effector-memory-t-cells-ear-after-induction-adoptive

Research

JoVE Journal

Biology

10.2K 조회수.  University of California, Irvine (UCI). 여기서 우리는 쥐의 귀에 지연 유형 과민성 (DTH) 반응의 진행 상황을 유도하고 기록하는 방법을 보여줍니다. 이것은 이펙터 / 메모리 T 세포 응답의 2 광자 이미징을위한 쥐의 귀 조직의 준비 시범옵니다.

비디오: Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

쥐에서 Active 지연 유형 과민성 (DTH)의 유도 및 모니터링

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

연구

생물학

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

고양이의 입양 지연 유형 과민성의 유도 및 모니터링

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

차 문화와 Corti의 Murine 오르간의 플라스미드 Electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic 분석 : 자기편 셀

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&amp;#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

디지털 Immunofluorescent 현미경을 사용하여 쥐 Pial Arterioles에서 에스 트로겐 수용체 - α의 영상

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

이미징 로컬 CA<sup&gt; 2 +</sup&gt; 배양 된 포유 동물 세포에서 신호

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

1차 마우스 망막 색소 상피 세포의 효율적인 해부 및 배양

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...