CRC Flow Cytometry Core Facility Knut och Alice Wallenbergs Stiftelse

Transcription

1 CRC Flow Cytometry Core Facility CRC Flow Cytometry Core Facility Knut och Alice Wallenbergs Stiftelse

2 CRC Flow Cytometry Core facility Instruments: 1. FACSCalibur: analyser, 6 parameters, 4 colors (BD), 2. CYAN ADP: analyser, 11 parameters, 9 colors (DAKO) 3. FACSAria: analyser and high-speed cell sorter 15 parameters, 13 colors (BD) 4. AutoMACS: automated magnetic cell sorter (enrichment) 5. LUMINEX: multiplex bead assays

3 Core facility Instruments: available 1. FACSCalibur: 2 lasers, 4 colors Operator independent Easy to learn No service contract

4 Core facility Instruments: available 2. CYAN ADP: 3 lasers, 9 colors Operator independent Easy to learn 1 year service contract

5 Core facility Instruments: available 3. FACSAria: 4 lasers, 13 colors 4 way cell sorting Operator dependent experience and training necessary 5 yrs service contract

6 Core facility Instruments: available 4. AutoMACS: magnetic negative and positive enrichments 2 column for sensitive sorting Operator independent No service contract

7 Core facility Instruments: available 5. Luminex platform: Beads based assay Multiparametric, theoretically up to 100 analytes in a single sample Low starting volume (20 ul) Operator independent Service contract 1 year

8 CRC Flow Cytometry Core Facility Models of running 1. Open usage 2. User club 3. User club and pay per service 4. Pay per Service

9 What can Flow Cytometry Do? Enumerate particles in suspension Determine biologicals from non-biologicals Separate live from dead particles Evaluate 10 5 to 10 6 particles in less than 1 min Measure particle-scatter as well as innate fluorescence or 2 o fluorescence Sort single particles for subsequent analysis

10 What are the principles? Light scattered by a laser Specific fluorescence detection Hydrodynamically focused stream of particles Electrostatic particle separation for sorting Multivariate data analysis capability

11 Definitions Flow Cytometry Measuring properties of cells in flow Flow Sorting Sorting (separating) cells based on properties measured in flow Also called Fluorescence-Activated Cell Sorting (FACS) but this term does refer to sorting, not analysis. FACS is frequently misused. Saying FACS when you mean flow cytometry is incorrect!!

12 Technical Components Illumination Sources Detection Systems Fluidics Sorting Data Acquisition Data Analysis Biological Systems

13 Flow Cell Injector Tip Sheath fluid Fluorescence signals Focused laser beam

14 Forward Angle Light Scatter Laser FALS Sensor

15 90 Degree Light Scatter Laser FALS Sensor 90LS Sensor

16 Fluorescence Detectors Laser FALS Sensor Freq Fluorescence Fluorescence detector (PMT3, PMT4 etc.)

17 Flow Cytometry Optics PMT 4 Flow cell Dichroic Filters PMT 1 PMT 2 Bandpass Filters Laser PMT 3

18 Common Laser Lines nm 400 nm 500 nm 600 nm 700 nm PE-TR Conj. Texas Red PI Ethidium PE FITC cis-parinaric acid

19 Gating strategy: morphology Lymphocyte gate R1 3. Granulocytes SSC 1. Lymphocytes R1 2. Monocytes FSC

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21 Multicolor analysis FITC PE PerCP or PE-Cy5.5 PE-Cy7 and more egfp PI PE-TxR PerCP PE-Cy5 488 nm 633 nm UV APC Indo-1 (Ca 2+ - flux) APC-Cy5.5 APC-Cy7 Hoechst DAPI Alexa 430 Cascade Blue ecfp 407 nm

22 Multi-color studies generate a lot of data 3 color 4 color 5 color

23 Cell sorting Cell of interest FLUORESCENT ACTIVATED CELL SORTING (FACS) Laser beam passes through one cell Cells in suspension are tagged with fluorescent markers specific for each cell type to be sorted Labelled cells are sent under pressure through a small nozzle and pass through an electric field A cell generates a negative charge if it fluoresces and a positive charge if it does not Sorted cell

24 CRC Flow Cytometry Core Facility Flow Cytometric analysis of the cell cycle

25 The Cell Cycle G2 M S G1 G0 Quiescent cells

26 Definitions & Terms Ploidy related to the number of chromosomes in a cell Haploid: Number of chromosomes in a gamete (germ cell) is called the HAPLOID number for that particular species Diploid: The number of cells in a somatic cell for a particular species

27 Definitions & Terms Hyperdiploid: greater than the normal 2n number of chromosomes Hypodiploid: Less than the normal 2n number of chromosomes DNA Tetraploidy: Containing double the number of chromosomes

28 DNA Analysis Side Scatter FALS Forward Scatter Forward Scatter Forward Scatter FLUORESCENCE Fluorescence Fluorescence

29 A typical DNA Histogram G 0 -G 1 S G 2 -M # of Events Fluorescence Intensity

30 CRC Flow Cytometry Core Facility Flow Cytometric analysis of Apoptosis

31 Lymphocytes Apoptosis Normal healthy lymphocyte Apoptotic lymphocyte

32 Detection of Apoptosis using cell cycle analysis

33 Detection of Apoptosis by flow cytometry A B Apoptotic cells (hypodiploidy) Apoptotic cells G0/G1 Cell numbers Cell numbers S phase G2/M TUNEL (FITC-dUTP) PI-Fluorescence A: TUNEL assay B: Cell cycle analysis

34 Annexin V staining

35 CRC Flow Cytometry Core Facility Flow cytometric analysis of cell proliferation

36 Measurement of T cells proliferation by CFSE staining and analysis of cell division: a flow cytometric approach CFSE: Carboxy-fluorescein diacetate, succinimidyl ester CFSE is a fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties. CFSE diffuses freely into cells and intracellular esterases cleave the acetate groups converting it to a fluorescent, membrane impermeant dye. The dye is not transferred to adjacent cells. CFSE is retained by the cell in the cytoplasm and binds the amino group of cytoplasmic proteins and does not adversely affect cellular function. During each round of cell division, CFSE labeled proteins are equally distributed between the doughter cells thus the relative intensity of the dye is decreased by half.

37 T cell proliferation by Flow cytometry and CFSE staining Cell divisions CFSE

38 CRC Flow Cytometry Core Facility Detection and characterization of Ag-specific T cells by flow cytometry

39 T cell response to allergen detected by Flow cytometry and CFSE

40 Cytokine secretion assay, CSA A general approach to detect low-frequency antigen specific T cells directly from peripheral blood based on in vitro antigeninduced secretion of cytokines and cellular affinity matrix technology

41 CRC Flow Cytometry Core Facility Flow Cytometric analysis of cellular function

42 Cellular Functions Cell Viability Phagocytosis Oxidative Reactions Superoxide Hydrogen Peroxide Nitric Oxide Glutathione levels Ionic Flux Determinations Calcium Intracellular ph Membrane Potential Membrane Polarization Lipid Peroxidation

43 Functional Assays intracellular ph intracellular calcium intracellular glutathione oxidative burst phagocytosis

44 PI - Cell Viability How the assay works: PI cannot normally cross the cell membrane If the PI penetrates the cell membrane, it is assumed to be damaged Cells that are brightly fluorescent with the PI are damaged or dead Viable Cell Damaged Cell PI PI PI PI PI PI PI PI PI PI PI PI PI PI

45 Phagocytosis Uptake of Fluorescent labeled particles Determination of intracellular or extracellular state of particles How the assay works: Particles or cells are labeled with a fluorescent probe The cells and particles are mixed so phagocytosis takes place The cells are mixed with a fluorescent absorber to remove fluorescence from membrane bound particles The remaining fluorescence represents internal particles FITC-Labeled Bacteria

46 Oxidative Burst generation of toxic oxygen species by phagocytic cells superoxide anion measured with hydroethidine hydrogen peroxide measured with 2,7 -dichlorofluorescin diacetatem (DCFH-DA)

47 Hydroethidine HE EB H 2 N NH 2 O 2 - H 2 N NH 2 H N N+ Br - CH2 CH 3 CH 2 CH 3 NADPH Oxidase Phagocytic Vacuole NADPH NADP O 2 O 2 - HE O 2 -H 2 O 2 DCF OH - SOD H2 O 2 DCF Example: Neutrophil Oxidative Burst

48 Calcium Flux Flow Cytometry Image Cytometry Stimulation Time (Seconds) Ratio: intensity of 460nm / 405nm signals Time (seconds) Indo-1

49 Oxonol Probes How the assay works: Membrane Potential Cyanine Probes Carbocyanine dyes released into the surrounding media as cells depolarize Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs PMA Added fmlp Added Repolarized Cells Green Fluorescence Time (sec) Green Fluorescence Time (sec) Depolarized Cells

50 CRC Flow Cytometry Core Facility Cell signaling analysis by flow cytometry: phosphoflow

51 Dendritic cell signalling pathways