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생명과학보고서_Immunostaining (아주대전공실험2)

아주대학교 전공실험2 만점 보고서입니다
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최초등록일 2023.09.13 최종저작일 2022.10
immunostaining 생명과학 생물학 생물학실험
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생명과학보고서_Immunostaining (아주대전공실험2)
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  • 미리보기

    소개

    아주대학교 전공실험2 만점 보고서입니다

    목차

    1. 실험목적
    2. 실험원리
    3. 실험재료 및 방법
    4. 참고문헌

    본문내용

    1. 실험목적
    본 실험은 cell culture를 통해 세포를 배양하여 counting해보고 적정 confluence에 맞춰 immunostaining을 통해 세포의 상태를 시각적으로 직접 관찰해보기 위해 수행되었다.

    2. 실험원리
    Cell culture란 다세포 생물체로부터 분리한 세포를 protease 등의 처리로 단세포로 분리하여 배양하는 과정이다. 우선 생체조직을 무균적으로 선발하여 trypsin 등의 소화효소로 처리한 후 단세포로 분리하여 초대배양을 한다. 또 계대 중인 세포계나 세포주를 같은 효소처리로 분산시켜 얻어낸 단세포를 증식배지에 이식, 접종하여 다음의 계대배양을 한다. Culture condition은 세포 type에 따라 매우 다양하지만 기본 환경은 필수 영양소(탄수화물, 아미노산, 비타민), serum(호르몬, growth factor), 가스(산소, 이산화탄소)를 공급하고 물리화학적 환경(pH, 온도)이 조절되어야 한다. pH 지시약으로서 media에 첨가되는 페놀레드는 pH 6.8~8.4 변색역을 가지며 산성색은 황색, 염기성색은 적색이다. 세포배양에서 세포를 키우는 기본 시스템이 두가지 있다.
    대부분의 세포는 solid or semi-solid substrate에 부착된 상태로 배양(adherent culture)되어야 하지만 일부는 media에 부유한 상태로 배양(suspension culture)될 수 있다. 표 1을 참고하면 각 방법이 적절하고 활용되는 실험은 각기 다름을 알 수 있다. Adherent culture에서는 세포가 surface에서 떼어져야 하는데, 그 방법으로는 흔들거나 긁어내는 기계적 방법과 trypsin과 같은 효소를 이용하는 방법이 있다. 표 2를 보면, 세포의 종류, 부착 강도에 따라 방법을 선택하면 됨을 알 수 있다.

    참고자료

    · Gagnieur, L., Cheval, J., Gratigny, M., Hébert, C., Muth, E., Dumarest, M., & Eloit, M. Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture. Biologicals, 42(3), (2014)., pp.145-152.
    · Invitrogen, Gibco. The Molecular Probes handbook: A guide to fluorescent probes and labeling technologies. Thermo Fish. Sci. (2010). pp.833-857
    · Invitrogen, Gibco. (2016). Cell culture basics handbook. Thermo Fish. Sci. pp.2-30
    · Perdue, T. D., & Parthasarathy, M. V. In situ localization of F-actin in pollen tubes. European journal of cell biology, 39(1), (1985). pp.13-20.
    · 박수용. 돼지 난자의 체외성숙에서 Bisphenol A에 의해 유도된 미토콘드리아 유래 활성산소 조절에 관한 연구. 국내석사학위논문. 대구대학교. 경상북도. (2016). p.9
    · 오충훈, 홍승희. 사람의 암 세포주에서 녹색 형광단백질 유전자의 발현 양상. 대한구강악안면 병리학 회지. 34(5), (2010). p.256
    · 이규엽,. 마우스 와우의 형태학적 검사방법. 대한이비인후과학회지-두경부외과학, 54(7), (2011). pp.445-453.

  • AI와 토픽 톺아보기

    • 1. Cell Culture
      Cell culture is a fundamental technique in the field of biology and biomedical research. It involves the growth and maintenance of cells in a controlled laboratory environment, allowing for the study of cellular processes, disease mechanisms, and the development of new therapies. Cell culture provides a valuable tool for understanding the behavior and characteristics of different cell types, as well as their responses to various stimuli and treatments. The ability to cultivate cells in vitro has been instrumental in advancing our knowledge of cellular biology, enabling researchers to conduct experiments that would be difficult or impossible to perform in living organisms. Cell culture techniques have been widely adopted across various disciplines, from basic research to drug discovery and development, and have played a crucial role in advancing our understanding of human health and disease.
    • 2. Adherent Culture and Suspension Culture
      Adherent culture and suspension culture are two distinct approaches in cell culture, each with its own advantages and applications. Adherent culture involves the growth of cells that require a solid surface or substrate to attach and proliferate, such as tissue culture plates or flasks. This method is commonly used for the culture of many mammalian cell lines, as it mimics the natural environment of these cells within the body. Suspension culture, on the other hand, involves the growth of cells that can freely float in the culture medium without the need for attachment. This technique is often employed for the culture of cells that naturally grow in suspension, such as certain types of immune cells or transformed cell lines. The choice between adherent and suspension culture depends on the specific cell type, the experimental objectives, and the desired cellular characteristics. Both approaches have their unique strengths and are essential tools in the field of cell biology, enabling researchers to study and manipulate cells in diverse experimental settings.
    • 3. Hemocytometer
      The hemocytometer is a crucial tool in cell culture and cell biology, used for the accurate counting and quantification of cells. This device consists of a specialized glass slide with a defined grid pattern and depth, allowing for the precise measurement of cell density in a sample. By loading a small volume of the cell suspension onto the hemocytometer and counting the cells within a specific grid area, researchers can determine the number of cells per milliliter or per unit volume. This information is essential for various applications, such as seeding cells at the appropriate density, monitoring cell growth and proliferation, and ensuring consistent cell numbers for downstream experiments. The hemocytometer's ability to provide reliable and reproducible cell counts makes it an indispensable tool in cell culture, hematology, and a wide range of biological and biomedical research fields. Its simplicity, accuracy, and widespread use have solidified the hemocytometer's status as a fundamental instrument in the study of cells and cellular processes.
    • 4. Cell Confluence
      Cell confluence is a critical parameter in cell culture, referring to the percentage of the available growth surface area that is covered by adherent cells. Monitoring and maintaining the appropriate level of cell confluence is essential for the health and behavior of cultured cells, as well as the success of various experimental procedures. At low confluence, cells may not have sufficient cell-cell interactions or access to nutrients, leading to suboptimal growth and proliferation. Conversely, high confluence can result in contact inhibition, where cells stop dividing and may undergo changes in their morphology and gene expression. Maintaining cells at the optimal confluence level, typically between 70-90%, ensures that they are in a healthy, actively proliferating state, ready for passaging, experimentation, or further manipulation. Careful monitoring and control of cell confluence are crucial for maintaining consistent and reliable cell culture conditions, enabling researchers to obtain meaningful and reproducible results from their experiments.
    • 5. Immunocytochemistry
      Immunocytochemistry (ICC) is a powerful technique in cell biology that allows for the visualization and localization of specific proteins or molecules within individual cells. By using antibodies that bind to target proteins, ICC enables researchers to study the expression, distribution, and subcellular localization of these molecules in cultured cells. This method is particularly valuable for understanding cellular processes, signaling pathways, and the organization of cellular structures. ICC can be combined with various imaging techniques, such as fluorescence microscopy, to provide high-resolution, multicolor images that reveal the spatial relationships between different cellular components. The ability to precisely detect and localize proteins of interest within the cellular context has made immunocytochemistry an indispensable tool in cell biology research, contributing to our understanding of cellular function, disease mechanisms, and the development of new diagnostic and therapeutic approaches.
    • 6. Fluorescence Microscopy
      Fluorescence microscopy is a powerful imaging technique that has revolutionized the field of cell biology and biomedical research. By utilizing fluorescent probes or labels that bind to specific cellular components, fluorescence microscopy allows for the visualization and study of the intricate structures and dynamics within living cells. This method enables researchers to observe and analyze the localization, interactions, and movements of proteins, organelles, and other biomolecules in real-time, providing unprecedented insights into cellular processes. The versatility of fluorescence microscopy, with its ability to employ a wide range of fluorescent dyes and genetically encoded fluorescent proteins, has made it a crucial tool for investigating diverse biological phenomena, from cell signaling and trafficking to gene expression and cell division. The continuous advancements in fluorescence microscopy, including super-resolution techniques and live-cell imaging, have further expanded the capabilities of this technology, allowing researchers to push the boundaries of our understanding of cellular biology and paving the way for groundbreaking discoveries in the life sciences.
    • 7. Fluorescent Labeling Techniques
      Fluorescent labeling techniques are essential tools in cell biology and biomedical research, enabling the visualization and tracking of specific biomolecules, cellular structures, and dynamic processes within living cells. These techniques involve the use of fluorescent dyes, proteins, or other labels that can be selectively attached to or incorporated into the target of interest, such as proteins, nucleic acids, or organelles. The wide range of available fluorescent probes, each with unique spectral properties and labeling strategies, allows researchers to simultaneously visualize multiple cellular components and study their interactions and spatial relationships. Fluorescent labeling techniques, combined with advanced microscopy methods, have revolutionized our understanding of cellular organization, signaling pathways, and the complex dynamics of biological systems. From immunocytochemistry and live-cell imaging to super-resolution microscopy and single-molecule tracking, these labeling approaches have become indispensable for unraveling the intricate workings of cells and tissues, ultimately contributing to advancements in fields like cell biology, developmental biology, and disease research.
    • 8. Reagents Used in Immunocytochemistry
      Immunocytochemistry (ICC) relies on a variety of specialized reagents to enable the specific detection and localization of target proteins or molecules within cells. These reagents include primary antibodies, which bind to the protein of interest, and secondary antibodies, which are conjugated to fluorescent dyes or enzymes and bind to the primary antibodies, amplifying the signal. Other essential reagents in ICC include fixatives, permeabilization agents, blocking solutions, and mounting media, all of which play crucial roles in preserving cellular structure, enhancing antibody accessibility, and preparing the samples for imaging. The careful selection and optimization of these reagents are crucial for obtaining high-quality, specific, and reproducible results in ICC experiments. The availability of a wide range of commercially produced, well-characterized reagents has greatly facilitated the widespread adoption and standardization of ICC techniques, enabling researchers to investigate the spatial distribution and expression patterns of diverse cellular components with confidence and reliability. The continuous development and refinement of these specialized reagents have been instrumental in advancing our understanding of cellular biology and driving progress in various fields of biomedical research.
    • 9. DAPI and Rhodamine-Phalloidin Staining
      DAPI (4',6-diamidino-2-phenylindole) and rhodamine-phalloidin are two commonly used fluorescent stains in cell biology and microscopy. DAPI is a DNA-binding dye that emits blue fluorescence when bound to the minor groove of double-stranded DNA, allowing for the visualization of cell nuclei. Rhodamine-phalloidin, on the other hand, is a fluorescent derivative of the natural compound phalloidin, which binds specifically to filamentous actin (F-actin) in the cytoskeleton. The combination of DAPI and rhodamine-phalloidin staining provides a powerful tool for studying the organization and dynamics of cellular structures, as it enables the simultaneous visualization of the nucleus and the actin cytoskeleton within the same cell. This dual-staining approach is widely used in a variety of applications, such as cell morphology analysis, cell cycle studies, and the assessment of cytoskeletal changes in response to various stimuli or treatments. The ease of use, specificity, and compatibility with common fluorescence microscopy techniques have made DAPI and rhodamine-phalloidin staining indispensable in the field of cell biology, contributing to our understanding of fundamental cellular processes and the development of new research methodologies.

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