Profundity
Integer organ Cell Molecule Gene
Progress of Immunological Techniques
Classical Immunological Techniques：
Ag-Ab reaction Immuno-labelling techniques Cell detection techniques Cytokine detection techniques Molecular biological techniques Cell sorting Surface molecule detection techniques
Research type
Interpreting Multiparameter Data
Single parameter (univariate) can be displayed as a histogram. Dual parameter (bivariate) data can be displayed in two dimensions using dot, density or contour plots.
Advantages: Fast, Numerous, Sensitive, Accurate, Quantitative Applications: Scientific research Clinical Analysis
Flow Cytometry and Cell Sorting : An Introduction
The Cell Cycle
G1 S phase M
Events
DNA content by flow cytometry
G1
G2
G0/1 2 1 S G2/M
1
DNA content Deconvolution into cell cycle phases
Measurement of sub G0/1 apoptotic particles
荧光信号 (fluorescence signals)
特异荧光
自发荧光
特异荧光
Hydrodynamic Focusing
Sheath fluid
Laminar Coaxial Flow
The Bernoulli Effect Direction of flow Lower pressure Velocity Gradient
Typical density : 105 - 107 cells / ml. up to 108 cells / ml on the MoFlo
Acquisition speeds : up to 10,000 events/sec typical 3,000 up to 150,000 on the MoFlo Sorting speeds: up to 100,000/sec typically 30,000 30,000/sec ~ 108 / hour
Development and Application of Immunological Techniques
Shi Yanchun
Research Center of Molecular Biology
Inner Mongolia Medical College
Preface
Development of Immunology
Three and more parameters are usually displayed as sets of histograms and bivariate plots.
Data Type of FACS
X轴：代表荧光信号或
散射光信号的强度，用 “通道数”表示，可以 与光强度之间线性关系 或对数关系
Intrinsic : DNA content, DNA composition, DNA synthesis chromatin structure, RNA, protein, sulphydryl groups, antigens(surface, cytoplaspic & nuclear), lectin binding sites, cytoskeletal components, membrane structure (potential, permeability & fluidity),enzyme activity, endocytosis, surface charge, receptors, bound and free calcium, apoptosis, necrosis, pH, drug kinetics, etc., etc.
检测信号（二）－－荧光信号
激光光束(通常λ488nm)与细胞正交时，可产生两种荧光信号 细胞自发荧光：激光照射下细胞自身发出的微弱荧光信号 荧光素发出的荧光：特异标记细胞的荧光素发出的荧光信号 常用的荧光染料、单抗标记的荧光素： 碘化丙锭(propidium iodide, PI) －－－－ 藻红蛋白(phycoerythrin, PE)－－－－－ 异硫氰酸荧光素(fluorescein isthiocyanate, FITC) －－ 藻红蛋白-花青素(PE-Cy5) －－－－－－
Advance Immunological Techniques
Flow Cytometry, FCM
Magnetic Activated Cell Sorting, MACS
Immunoblotting/Western blot
Flow Cytometry
流式细胞术（Flow Cytometry，FCM） 是借助荧光激活细胞分类仪( fluorescence-activated cell sorter, FACS )对处在液流中的荧光抗体标记的细胞或其他生 物微粒(如细菌等）逐个进行多参数的快速定量分析和分选的 技术。
A Recent Example
The application of multiparametric flow cytometry to measuring rates of proliferation within the different haematopoietic lineages found in the bone marrow.
Progress of Immunological Techniques
Development of Immunology
Extent
Anti-infectious immunity Cross-discipline Reproductive Immunology Immunophysiology Immunopathology Immunotherapeutics Surroundings Immunology Immunopsychology
rHBsAg +CpG ODN %G0-G1: 81.15 %G2-M: 2.17 %S: 16.68 %Apoptosis: 20.83
Vaccine %G0-G1: 76.17 %G2-M: 2.21 %S: 21.61 %Apoptosis: 39.99
Vaccine +CpG %G0-G1: 77.27 %G2-M: 0.78 %S: 21.95 %Apoptosis: 29.30
检测信号（一）－－散射光
它反映细胞的物理特性,根据前、侧向散射光，可 以把不同类型的细胞群加以区分。
前向角散射(forward
scatter, FSC) :反应细胞的
相对大小和截面积的大 小
侧向角散射(side scatter,
SSC):代表细胞的颗粒度
和精细结构的变化
全血细胞流式FSC-SSC图
Cell cycle of splenocytes by FACS
Applications of Flow Cytometry
Cellular parameters measurable by flow cytometry.
Extrinsic : Size, shape, cytoplasmic granularity, autofluorescence and pigmentation.
假三维图 ( Pseudo 3D Plot ) 三维图( 3D Plot )
Pseudo 3D Plot
3D Plot
Control %G0-G1: 83.22 %G2-M: 0.04 %S: 16.74 %Apoptosis: 20.10
rHBsAg %G0-G1: 72.82 %G2-M: 3.72 %S: 23.46 %Apoptosis: 43.18
1970’s: 1991 : 1996 : 1st FACS The first commercial machine Haematology, Medicine, Biochemistry Biochemistry Pathology Upgrades to older machines High speed cell sorter
FACScans FACSCali1 : 2nd FACSCaliburs
Cytomation MoFlo
Cell Sorting by FACS
Properties of FSC and SSC
SSC
Incident Light
FSC Forward Scatter ( FSC ) －Diffracted light －Related to cell surface area －Detected along axis of incident light in the forward direction Side Scatter ( SSC ) －Reflected and refracted light －Related to cell granularity and complexity －Detected at 90° to the laser beam