Introduction to Flow Cytometry. -- BD FACSCanto II TM. Daisy Kuo Application Specialist BDBiosciences

Transcription

1 Introduction to Flow Cytometry -- BD FACSCanto II TM Daisy Kuo Application Specialist 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 BD FACSCanto II TM 10

11 Subsystems Fluidics To introduce and focus the cells for interrogation. Optics To generate and collect the light signals. Electronics To convert the optical signals to proportional digital signals, process the signals, and communicate with the computer. 11

12 Sample Flow Excitation Lasers Hydrodynamic Focusing sheath sample sheath 12

13 Optics Excitation optics Lasers Lenses to shape and focus the laser beam Collection optics A collection lens to collect light emitted from the article-laser beam interaction A system of optical mirrors and filters to route specified wavelengths of emitted light to designated optical detectors 13

14 Fluorochrome Spectra 14

15 Excitation Optics Spatially separated laser beams lower the possibility of fluorescence spillover 15

16 Collection Optics 16

17 Optics-- Configuration Other Fluorochrome GFP PI PI, PE-Cy5.5, 7-AAD Alexa Fluor 633 DAPI, Hoechst Dye Cascade Blue 17

18 Electronics PMTs and preamps convert photons to voltage pulses. Analog-to-digital converters translate analog signals to proportional digital signals. Compute area and height for each pulse. Perform compensation and calculate ratios and width. An embedded computer interfaces with the computer workstation for data transfer. 18

19 Creation of a Voltage Pulse Laser Volts Pulse Height Pulse Width Time (µs) 19

20 20 Analog-to-Digital Converter Baseline 16,364 Time 937 1,985 7,650 12,420 Height Digitized values

21 Quantification of a Voltage Pulse 21

22 Data Storage Event 1 Event 2 Event 3 Time List-Mode Data FSC SSC FITC PE , , ,271 30,621 22,688 6,189 39,271 FITC FITC 89 PE ,621 PE 22

23 Review Time Time Time Data Processing SSC PE FSC Time FITC APC Time PerCP-Cy5.5 Time 23

24 Application Examples

25 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 25

26 Phenotype Analysis Ligand Receptor Adhesion molecule etc 26

27 Lymphocyte Immunophenotyping Peripheral White Blood Cells CD45 + Monocytes Monocytes Lymphocytes Granulocytes Neutrophils T B NK T Helper CD3 + CD4 + CD3 + T Cytotoxic CD3 + CD8 + CD3 - CD19 + CD3 - CD16 + CD56 + Basophils Eosinophils 27

28 28

29 Intracellular Analysis Permeabilizing solution Cytokine Enzyme signal transduction molecule etc. Fixation solution 29

30 Cytokine Detection Picture From 30

31 APC LPS Ionomycin PHA Super Ag MHC T cell Con A TCR APC MHC CD28 CD3 Stimulation Intracellular Staining To enhance the accumulation of intracellular cytokines. Secretion stop Monensin: Cytokines accumulate in the ER Brefeldin A: in Golgi complex. (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 Permeabilisation Saponin (permeablisation buffer). Fixation 31

32 Combination of Cell Surface and Cytoplasmic Staining Th1/Th2/Th17 Phenotyping Kit 32

33 Signal Transduction 33

34 Intracellular Staining in Activated Lysed Whole Blood 34

35 DNA Analysis Ethanol Detergent Nucleic Acid Dye 35

36 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 36

37 Apoptosis (Sub G1) 37

38 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) 38

39 Annexin V Assay Annexin V-FITC conjugate C a++ C a++ C a++ C a++ Apoptosis Plasma membrane Externalization of phosphatidylserine Cytoplasm Cytoplasm 39

40 Annexin V/PI Double Staining Bordón et al. Radiation Oncology :58 40

41 Mitochondria Membrane Potential (JC-1) 41

42 Cytometric Beads Array (CBA) Single Step Incubation or Two-Step Incubation 42

43 Beads Provide a Flexible Platform Multiple sizes Different fluorescence intensities Different colors with different intensities 43

44 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) 44

45 Proteins Measured A. Interleukin (IL)-2 B. IL-4 C. IL-5 D. IL-10 E. Tumor Necrosis Factor- F. Interferon- 45

46 Cytometry Beads Array (CBA) 46

47 Standard Curves 47

48 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 48

49 CBA Functional Beads Can be conjugated with any Ab 49

50 Fluorescent Cell Barcoding (FCB) 50

51 Fluorescent Cell Barcoding (FCB) 51

52 Fluorescent Cell Barcoding (FCB) 52

53 Fluorescent Cell Barcoding (FCB) 53

54 BD Phosflow Violet Fluorescent Cell Barcoding Kit 54

55 Fluorescence Protein Application: GPS (Global Protein Stability) Profiling Control of protein turnover serves as a rapid mechanism for the activation or inhibition of signaling pathways when cells respond to environmental changes. Regulated degradation of cancer-related proteins play important roles in cellular transformation, and multiple components of the proteolysis system are directly involved in human diseases. Global protein stability profiling in mammalian cells. Science 332,

56 Fluorescence Protein Application: GPS (Global Protein Stability) Profiling Global protein stability profiling in mammalian cells. Science 332,

57 Fluorescence Protein Application: GPS (Global Protein Stability) Profiling Global protein stability profiling in mammalian cells. Science 332,

58 Thank You!