생명과학보고서_Immunostaining (아주대전공실험2)
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생명과학보고서_Immunostaining (아주대전공실험2)
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2023.09.14
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  • 1. Cell Culture
    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.
  • 2. Adherent Culture and Suspension Culture
    대부분의 세포는 solid or semi-solid substrate에 부착된 상태로 배양(adherent culture)되어야 하지만 일부는 media에 부유한 상태로 배양(suspension culture)될 수 있다. Adherent culture에서는 세포가 surface에서 떼어져야 하는데, 그 방법으로는 흔들거나 긁어내는 기계적 방법과 trypsin과 같은 효소를 이용하는 방법이 있다.
  • 3. Hemocytometer
    혈구 계수기는 혈구수 또는 그 밖의 입자수를 측정하는 계량기이다. 깊이 0.1mm, 1mm2 구획 9개로 구성되어 있고 1mm2 한 칸의 volume은 10-4ml이다. 따라서 count한 세포 수의 계산은 sample 1ml당 1mm2 한 칸에 든 평균 세포 수에 104를 곱한 것만큼 있다고 생각하면 된다.
  • 4. Cell Confluence
    cell culture에서, confluence는 adherent cell에 의해 덮인 dish의 표면적 비율을 의미한다. 예를 들어 50% confluence는 표면의 절반가량 덮여 있다는 것이고, 100% confluence는 표면이 다 덮여 더 이상 세포가 monolayer로 자랄 공간이 없다는 것을 의미한다. 세포를 seeding할 때는 70~80% confluence를 갖도록 seeding해주도록 한다.
  • 5. Immunocytochemistry
    Immunocytochemistry는 특이성을 지닌 1차 항체를 사용하여 세포에 있는 특정 단백질이나 antigen의 위치를 해부학적으로 시각화하는 실험 기법이다. 항원-항체 반응을 이용하여서 매우 specific하고 실제 위치를 시각화할 수 있다는 장점을 가진다. 1차 항체는 형광물질을 가진 2차 항체에 결합할 때 형광 현미경에서 단백질을 시각화한다.
  • 6. Fluorescence Microscopy
    Fluorescence microscopy은 특정 파장의 빛을 시료에 쪼인 후 시료 내에 분포된 형광 물질로부터 방출되는 형광을 측정하여 형광 이미지를 얻는 방법이다. 이때 형광 현미경의 대물렌즈는 빛을 시료에 쪼이고 동시에 시료에서 방출되는 형광을 광검출기로 모으는 역할을 한다.
  • 7. Fluorescent Labeling Techniques
    형광염색에는 3가지 방법이 있다. 첫번째로, 관찰하고자 하는 물질(항원)에 직접 반응하여 형광을 내는 것이다. 두번째, 녹색형광단백질(green fluorescent protein, GFP)은 자외선을 조사하면 그 자체가 녹색(510nm)혹은 청색(395nm)의 형광을 발생한다. 세번째, 특이적 항체 결합을 이용한 면역형광법(면역염색법)이다.
  • 8. Reagents Used in Immunocytochemistry
    Trypsin은 protease로서 배양할 때 단세포로 분리하고, adherent cell을 surface에서 떼어 내기 위해 사용된다. EDTA는 세포가 바닥 또는 서로 붙을 때 필요한 Ca2+를 붙잡아서 그들 사이의 결합력을 약화시킨다. FBS (Fetal Bovine Serum)은 α1-antitrypsin과 같은 단백질분해효소 저해제를 가지고 있어 trypsin을 불활성화함으로서 그로 인한 세포 손상을 방지한다. Paraformaldehyde는 formaldehyde의 중합체로 항원결정부의 인식을 저해하지 않으므로 대부분의 면역형광법에서 가장 좋은 고정액이다.
  • 9. DAPI and Rhodamine-Phalloidin Staining
    DAPI (4′,6-diamidino-2-phenylindole)는 DNA의 A-T rich region에 붙는 형광염색물질이고, Rhodamine-phalloidin은 phalloidin이 cytoskeleton 중 actin filament의 F-actin에 특이적으로 결합할 때 rhodamine의 derivative인 TRITC가 580nm(주황색)에서 발광하는 것을 이용한 것으로, actin filament을 labeling하는 데 사용된다.
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  • 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.