Introduction to Flow Cytometry. -- BD FACSCanto II TM. Daisy Kuo Assistant Product Manager BDBiosciences

Transcription

1 Introduction to Flow Cytometry -- BD FACSCanto II TM Daisy Kuo Assistant Product Manager BDBiosciences

2 Outline Basic Concept of Flow Cytometry FACSCanto II System Introduction Application Examples 2

3 What is Flow Cytometry? Flow = Fluid Cyto = Cell Metry = Measurement A variety of measurements are made on cells, cell organelles, and other objects suspended in a liquid and flowing at rates of several thousands per second through a flow chamber. 3

4 Particle Size Detection range: 0.5~50um 4

5 What Can a Flow Cytometer Tell Us About a Cell? Its relative size (Forward Scatter FSC) Its relative granularity or internal complexity (Side Scatter SSC) Its relative fluorescence intensity 5

6 Scatter Light Laser FSC Sensor SSC Sensor 6

7 Lysed Whole Blood Side Scatter Neutrophils Forward Scatter Monocytes Lymphocytes 7

8 Fluorescence Light 8

9 Fluorescence 9

10 Applications Phenotype Analysis (Cell Surface Antigens/Markers) Intracellular Analysis -- Eg. Cytokines, Signal Transduction molecules etc. DNA Analysis -- Eg. Viability, Cell cycle, Apoptosis etc. Cell Fuction Analysis -- Eg. Free radicals, Ca 2+, Reporter genes etc. CBA (Cytometric Bead Array) Others 10

11 BD FACSCanto II TM : 2 Laser, 6 Color 11

12 Configuration Other Fluorochrome GFP PI PI, PE-Cy5.5, 7-AAD Alexa Fluor

13 Phenotype Analysis Ligand Receptor Adhesion molecule etc 13

14 Lymphocyte Immunophenotyping Peripheral White Blood Cells CD45 + Monocytes Granulocytes Monocytes Lymphocytes Neutrophils T B NK T Helper CD3 + CD3 - T Cytotoxic CD3 + CD4 + CD3 + CD8 + CD19 + CD3 - CD16 + CD56 + Basophils Eosinophils 14

15 15

16 Tools for Immunophenotyping

17 Surface Marker Screening 17

18 Intracellular Analysis Permeabilizing solution Cytokine Enzyme signal transduction molecule etc. Fixation solution 18

19 Cytokine Detection Picture From 19

20 LPS Ionomycin PHA T cell Con A CD28 CD3 Stimulation To enhance the accumulation of intracellular cytokines. Monensin: Cytokines accumulate in the ER Brefeldin A: in Golgi complex. Secretion stop (Brefeldin A or Monensin) Only in vitro To maintain structural integrity. Formaldehyde or glutaraldehyde Keep the protein structure and doesn't change the (accessibility of the) epitopes too much Intracellular Staining Permeabilisation Saponin (permeablisation buffer). Fixation 20

21 Combination of Cell Surface and Cytoplasmic Staining 21

22 Signal Transduction 22

23 Intracellular Staining in Activated Lysed Whole Blood 23

24 DNA Analysis Ethanol Detergent Nucleic Acid Dye 24

25 Cell Cycle Analysis G 2 M G 0 G 1 G 0 G 1 s C o u n t s G 2 M N 4N DNA content 25

26 Apoptosis (Sub G1) 26

27 Cell Function Analysis Membrane Potential (DiOC6, JC-1) Oxidative Metabolism (Free Radicals) Intracellular PH Value (Snarf-1) Ca++ Influx (Fluo-4/Fura Red, Indo-1) Phagocytosis Cell Proliferation (PI, BrdU, Intracellular Cyclins) Apoptosis (Annexin V, active Caspase-3) 27

28 Annexin V Assay Annexin V-FITC conjugate C a++ C a++ C a++ C a++ Apoptosis Plasma membrane Externalization of phosphatidylserine Cytoplasm Cytoplasm 28

29 Annexin V/PI Double Staining Bordón et al. Radiation Oncology :58 29

30 Cell Proliferation Divisions:

31 Cytometric Beads Array (CBA) Single Step Incubation or Two-Step Incubation 31

32 Beads Provide a Flexible Platform Multiple sizes Different fluorescence intensities Different colors with different intensities 32

33 Advantages of Bead-Based Immunoassays Analyze multiple analytes simultaneously Reduced sample volume requirements Reduced hands-on time by parallel analysis of samples Wide dynamic range of fluorescence detection (requires fewer sample dilutions) 33

34 Proteins Measured A. Interleukin (IL)-2 B. IL-4 C. IL-5 D. IL-10 E. Tumor Necrosis Factor- F. Interferon- 34

35 Cytometry Beads Array (CBA) 35

36 Standard Curves 36

37 CBA Flex Sets Open configuration (Up to 30 plex) Clustering based on Red and NIR fluorescence intensity Need to be used at dual-laser(488nm blue v.s 633nm red) instrument 37

38 CBA Functional Beads Can be conjugated with any Ab 38

39 Fluorescent Cell Barcoding (FCB) 39

40 BD Phosflow Violet Fluorescent Cell Barcoding Kit 40