1. 인큐베이터에서 계란을 제거합니다 <ol><li> 37 ° C 유지 60% 위에 설정된 상대 습도. </li><li> 계란을 제거, 계란 90 ° 회전 큰 기지가 수평으로 놓여 있도록. </li></ol> 2. 면봉 계란 소독을 <ol><li> 70 % 에탄올과 비 멸균 거즈의 스택을 포화. </li><li> 5 달걀까지 면봉에 2 ~ 3 조각을 사용합니다. 거즈가 더러워진 경우 폐기하십시오. </li></ol> 3. 준비 횐자위 제거 사이트 <ol><li> 그냥 알부민이 밖으로 그려진 것입니다 지역을 보호하기 위해 기본 왼쪽 3M 플라스틱 테이프 1 &quot;X 1&quot;조각을 잘라 장소. </li></ol> 4. 횐자위 제거 <ol><li> 테이프의 중간에 작은 구멍을 만들기 위해 가위의 요점을 사용합니다. </li><li> 18 게이지 1 인치 바늘과 10 CC 주사기를 사용하면 천천히 가위에 의해 만들어진 구멍을 통해 바늘을 드릴. </li><li> 계란의 바닥을 향해 45 ° C 각도에서 바늘을 아래로 드라이브. </li><li> 중앙쪽으로 바늘을 기울이기 및 횐자위의 3-4 ML을 그립니다. </li></ol> 5. 윈도우 <ol><li> 플라스틱 테이프 3 &quot;X 3&quot;조각을 잘라 계란의 상단에 맞게 그것을 스트레칭. 너무 열심히 끌어 않도록 조심하고, 계란의 수평 표면의 둥근 끝을 주변 광장의 모서리를 확장합니다. 그것이없이 주름과 달걀의 표면에 대한 꽉되도록 테이프를 당겨. </li><li> 날카로운 직선 4 &quot;해부 가위의 쌍을 사용하여 테이프가 위치했던 지역의 하단 중앙에 구멍을 비꼬아. 천천히 안쪽에 대한 도움말을 계속해야되는 계란에 가위의 낮은 블레이드 안내 쉘은. 기본 향해 칼날을 직접 천천히 껍질을 잘라 시작합니다. 시계 반대 방향으로 방식으로 진행 그냥 맨 중심에 도달하기 전에 중지.만이 작은 비트까지 가위를 제거하고 반대 방향으로가는 반복 계란 첨부. 난자가 수정된 있는지 확인 확인은. 창을 닫아 남아있다. </li></ol> 6. 폐쇄 재개와 계란을 씰링. <ol><li> 넓은 비닐 테이프 &quot;2분의 1의 길이&quot;2-3에 대한 절단하고 절단했던 구멍으로 다시 맞는 있도록 창을 종료. 테이프의 다른 한 X 1 &quot;조각을 가지고 계란이 말라 버린하는 구멍을 밀봉. 모든 조작을 할 수있는 계란을 재개하기 위해 포셉의 한 켤레를 사용. 당신은 인큐베이터에 달걀을 반환하는 준비가되면, 한 조각 잘라 창문을 봉인하고 계란의 전체 수평 표면을 충당하기 위해 충분히 큰 테이프. </li></ol>

<ol>
	<li>Cramer, K. S., Fraser, S. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Biol. 224, 138-151 (2000).</li><li>Cramer, K. S., Bermingham-McDonogh, O., Krull, C. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Biol. 269, 26-35 (2004).</li><li>Cramer, K. S., Cerretti, D. P., Siddiqui, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Biol. 295, 76-89 (2006).</li><li>Huffman, K. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Neurobiol. 67, 1655-1668 (2007).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Dyn. 229, 433-439 (2004).</li><li>Gerlach-Bank, L. M., Cleveland, A. R., Barald, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Dyn. 229, 219-230 (2004).</li></ol>

이 방법은 실험 조작에 따라 긴 생존 시간을 허용합니다. 예를 들어, 염료 라벨링 연구는 나중에 운명 매핑 연구 (1) E10 또는 생존을 허용했다. 또한, 그것은 유전자 발현 수준의 변경 및 나중에 발달 단계 (2-6)를 진행하는 데 필요한 개발을 제공 비켜 electroporation 연구에 사용되었습니다. 이 윈도우 기술은 배아에 조작 후 생존을 요구하는 절차와 함께 사용할 수 있습니다.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Rotating incubator</td>
<td>&nbsp;</td>
<td>Profi</td>
<td>&nbsp;</td>
<td>Maintain at 37&#176; Degrees with relative humidity set above 60%</td>
</tr>
<tr>
<td>70 % EtOH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>gauze</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3M plastic tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10cc syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1/egg
</td>
</tr>
<tr>
<td>18G needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1&#189; inch needle, 1/syringe</td>
</tr>
<tr>
<td>Dissection scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4" sharp straight blades</td>
</tr>
<tr>
<td>Fertilized Eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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windowing chicken eggs for developmental studies

개발의 연구는 크게 실험 모델로 여자의 배아를 사용하여 주었되었습니다. 배아의 접근의 용이성은 여자 퀘일 chimeras 및 초점 염료 라벨 등 여러 방법을 사용하여 세포의 운명을지도하기 위해 실험에 사용할 수 있습니다. 또한, 그것은 단백질 코팅 구슬과 비켜 electroporation을 사용하여 플라스미드 DNA의 도입의 배치를 포함한 여러 종류의 분자 perturbations에 대한 수 있습니다. 이러한 실험은 중앙과 주변 신경 시스템의 개발에 중요한 데이터를 굴복했습니다. 이러한 연구의 많은 경우, 그것은 달걀 껍질을 열고 배아의 성장을 perturbing없이 reclose 필요합니다. 배아는 달걀 껍질을 다시 열어 연속 발달 단계에서 검사를하실 수 있습니다. 이 문서에서, 닭고기 계란을 열 몇 가지 훌륭한 방법이 있지만 우리는 긴 생존 시간에 대한 최적화된 한 가지 방법을 보여줍니다. 이 방법에서, 계란은 측면에 달려 있으며 작은 창이 껍질에 상처입니다. 실험 절차 후, 쉘은 개발 기간 동안 계란을 커버하는 데 사용됩니다. 달걀 껍질을 overlying 투명한 플라스틱 테이프는 배아를 보호하고 부화 기간의 남은 기간 동안 수화를 유지하는 데 도움이됩니다. 이 방법은 배양이 이틀 동안에 처음 사용되었으며, 성숙 배아 세를 통해 생존을 허용하고 있습니다.

Watch this Scientific Journal Video about Windowing Chicken Eggs for Developmental Studies at JoVE.com

Windowing Chicken Eggs for Developmental Studies

배아의 접근의 용이성은 여자 퀘일 chimeras 및 초점 염료 라벨 등 여러 방법을 사용하여 세포의 운명을지도하기 위해 실험에 사용할 수 있습니다. 이 문서에서 우리는 긴 생존 시간에 최적화된있는 하나의 계란 준비 방법을 보여줍니다.

발달 연구를위한 윈도우 치킨 계란

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

windowing-chicken-eggs-for-developmental-studies

Research

JoVE Journal

Biology

27.8K 조회수.  University of California, Irvine (UCI). 배아의 접근의 용이성은 여자 퀘일 chimeras 및 초점 염료 라벨 등 여러 방법을 사용하여 세포의 운명을지도하기 위해 실험에 사용할 수 있습니다. 이 문서에서 우리는 긴 생존 시간에 최적화된있는 하나의 계란 준비 방법을 보여줍니다.

비디오: Windowing Chicken Eggs for Developmental Studies

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

CD1d에 의해 Glycolipid의 항원 프레 젠 테이션 및 NKT 세포 활성화의 치료 가능성 : 인터뷰

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

연구

생물학

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

인터뷰 : 단백질 폴딩과 Neurodegenerative 질병의 연구

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Xenopus laevis 여성의 난자를 구하기

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

치킨의 배아에 인간 줄기 세포의 Xenotransplantation

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

에 의한 이전 치킨 태아에 유전자 이체<em&gt; 전 비켜</em&gt; Electroporation

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

STA-PUT 속도의 침전을 사용하여 정자 형성 세포 유형의 분리

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

문제 해결 전략 현대 양적 형광 웨스턴 블로 팅에 가이드

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

세포 투과성의 Cys를 사용하여 포유 동물 세포에 직접 단백질 배달<sub&gt; 2</sub&gt; - 그의<sub&gt; 2</sub&gt; 아연 손가락 도메인

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.