[약대 실습 레포트] 면역학 실습 레포트_면역세포의 분리 확인(cell surface staining)

문서 내 토픽

1. Immunofluorescence Microscopy

형광 항체 현미경은 cell이나 tissue 내의 target protein을 detection하는 immunohisto chemistry 의 한 방법으로, enzyme을 사용하는 것이 아니라, fluorophore가 결합된 항체를 이용하여 형관 현미경을 이용해 target protein 위치를 detection하는 방법이다. 형광 dye는 특정 파장의 빛을 흡수하며 그 파장대에 따라 보색을 나타낸다. 형광항체법은 직접형광 항체법과 간접형광항체법이 있다.
2. CD4 T cell과 CD8 T cell

세포성 면역의 주요 세포인 T cell은 흉선에서 유래하는 림프구로, naive T cell은 항원과 반응하면 cytokine을 생성하며 증식이 일어나고 활성화된 T cell로 분화한다. 활성화된 T cell은 세포 표면에 있는 단백질 분자에 의해 구분되며, Tc cell은 CD8+, Th cell은 CD4+, Treg은 CD4+, CD25+이다.
3. 유세포 분석

유세포 분석기는 부유액 내의 개별세포를 세기 위해 레이저빔과 광탐지기를 이용한다. 개별 cell이 레이저빔을 통과할 때마다 빛이 탐지기로부터 비껴나가고 광탐지기에 이러한 빛의 차단이 기록된다. 어떤 cell의 세포 표면 항원에 형광물질이 부착된 항체가 와서 결합하면 레이저빔에 의해 흥분되고 빛을 방출하게 된다. 이렇게 방출된 빛은 레이저 빔과 수직으로 위치하는 제 2의 탐지기에 의해 기록된다.
4. 실험 결과 분석

실험 결과 분석을 통해 전체 splenocyte cell population 중 lymphocyte와 비슷한 크기의 cell들을 R2로 분리하고, 이에 해당하는 cell들만 dot plot에 보이게 analysis하였다. 각 튜브별로 T cell, CD4+ T cell, CD8+ T cell의 비율을 확인할 수 있었다.
5. Flow Cytometry

FACS는 분석법의 정식 이름이 아니라 BD Bioscience에서 개발한 flow cytometry와 cell sorting이 동시에 가능한 기기의 상표이다. FACS는 한줄기의 유체에 들어있는 미립자를 조사하는 실험 기법으로, 광학상 또는 전지거인 검출방법을 통해 각각의 세포의 물리적, 화학적 특징 등 동시적인 여러 요소의 분석이 가능하다.
6. Apoptosis 관찰

Flow cytometry 장비를 이용하여 cell의 apoptosis를 관찰할 수 있다. Annexin V staining, mt membrane potential의 변화 감지, caspase detection, PARP cleavage detection, BrdU staining 등을 통해 apoptosis 정도를 확인할 수 있다.
7. Hybridoma 기술

항체를 분비하는 B cell과 myeloma cell line을 fusion해서 hybridoma cell line을 만드는 원리와 방법을 조사하였다. 항원을 마우스에 주사하여 B cell의 수를 늘리고, 이 B cell과 myeloma cell을 융합시켜 hybridoma cell line을 만들 수 있다.

Easy AI와 토픽 톺아보기

1. Immunofluorescence Microscopy

Immunofluorescence microscopy is a powerful technique that allows researchers to visualize and localize specific proteins or molecules within cells or tissues. This method involves labeling target molecules with fluorescent dyes or antibodies, which can then be detected and imaged using a fluorescence microscope. The ability to precisely identify the spatial distribution and abundance of proteins of interest is crucial for understanding cellular processes, disease mechanisms, and the effects of various treatments or interventions. Immunofluorescence microscopy has been widely used in fields such as cell biology, immunology, neuroscience, and cancer research, providing valuable insights that would be difficult to obtain through other imaging techniques. As the technology continues to evolve, with advancements in fluorescent probes, microscope hardware, and image analysis software, immunofluorescence microscopy remains an indispensable tool for researchers seeking to unravel the complexities of biological systems.
2. Flow Cytometry

Flow cytometry is a powerful analytical technique that has revolutionized the field of cell biology and immunology. This technology allows for the rapid and quantitative analysis of multiple parameters of individual cells within a heterogeneous population. By passing cells through a laser beam and detecting the scattered light and fluorescent signals, flow cytometry can provide information on cell size, granularity, and the expression of specific surface or intracellular markers. This capability enables researchers to identify, enumerate, and characterize different cell types, such as immune cells, stem cells, and cancer cells, with high precision and sensitivity. Flow cytometry has become an indispensable tool for a wide range of applications, including immunophenotyping, cell sorting, cell cycle analysis, and the detection of rare cell populations. As the technology continues to evolve, with advancements in fluorescent probes, fluidics, and data analysis software, flow cytometry remains a crucial technique for advancing our understanding of complex biological systems and driving progress in fields like immunology, cancer research, and regenerative medicine.
3. Apoptosis Observation

Apoptosis, or programmed cell death, is a fundamental biological process that plays a crucial role in development, homeostasis, and the immune response. Observing and understanding the mechanisms of apoptosis is essential for researchers in fields such as cell biology, immunology, and cancer biology. Various techniques, including microscopy, flow cytometry, and biochemical assays, have been developed to detect and quantify apoptosis in cells and tissues. These methods allow researchers to identify the morphological changes associated with apoptosis, such as cell shrinkage, membrane blebbing, and nuclear fragmentation, as well as the activation of specific signaling pathways and the expression of apoptosis-related proteins. Accurate observation and analysis of apoptosis are crucial for understanding the regulation of cell death, the pathogenesis of diseases characterized by excessive or insufficient apoptosis (e.g., cancer, neurodegenerative disorders, autoimmune diseases), and the development of targeted therapies that modulate apoptotic pathways. As the field of apoptosis research continues to evolve, with the discovery of new molecular players and the refinement of experimental techniques, the ability to reliably observe and interpret apoptotic events remains a valuable skill for researchers across various biomedical disciplines.