Flow Cytometry. Marta Argenti, PhD student. Department of Biomedical Sciences Padua

Transcription

1 Flow Cytometry Marta Argenti, PhD student Department of Biomedical Sciences Padua

2 Flow ~ cells in motion Cyto ~ cell Metry ~ measure Physical properties: Flow Cytometry is the measurement of cells in a flow system - size - granularity, internal complexity Chemical properties: - fluorescence intensity (fluorescent probes and Ab)

3 Uses of Flow Cytometry Immunophenotyping DNA cell cycle/tumor ploidy Membrane potential Ion flux Cell viability Intracellular protein staining ph changes Cell tracking and proliferation Sorting Redox state Chromatin structure Total protein Lipids Surface charge Membrane fusion/runover Enzyme activity Oxidative metabolism Sulfhydryl groups/glutathione DNA synthesis DNA degradation Gene expression... and many others

4 To measure large numbers of single cells within a short period of time (tens of seconds to minutes). Simultaneous collection of the most diverse parameters of each cell. Reproducibility and statistical reliability. The heterogeneity of populations can be revealed and different subsets of cells identified and quantified. Selected cell populations can also be physically sorted for further study. It requires a suspension of single cells.

5 A flow cytometer need to combine: Fluidics Introduces and focuses the cells in suspension for interrogation. Optics Generates and collects light signals. Electronics Changes optical signals to electronic signals and digitize for computer analysis.

6 The Fluidics System

7 Hydrodynamic focusing The cells flow one-by-one into the cytometer to do single cell analysis.

8 flow cell Becton Dickinson FACSAria sample

9 A flow cytometer need to combine: Fluidics Introduces and focuses the cells in suspension for interrogation. Optics Generates and collects light signals. Electronics Changes optical signals to electronic signals and digitize for computer analysis.

10 Optics cells Fluorescence / L A S E R

11 L A S E R Optics cell Fluorescence / L A S E R Forward Scatter (FSC) Side Scatter (SSC) diffracted light, it is a measure of CELL SIZE refracted and reflected light, it is a measure of GRANULARITY AND INTERNAL COMPLEXITY

12 Optics FL1 SSC 488/10 530/30 FL2 585/42 FL4 661/16 90/10 Beam Splitter DM 560SP Fluorescence / Side Scatter DM 640LP 670LP Half Mirror Fluorescence Collection Lens FL3 Blue Laser 488 nm Beam Combiner Red Laser 635 nm. Focusing Lens Flow Cell 488/10 FSC (FACScalibur 4 colours) sample Forward Scatter

13 A flow cytometer need to combine: Fluidics Introduces and focuses the cells in suspension for interrogation. Optics Generates and collects light signals. Electronics Changes optical signals to electronic signals and digitize for computer analysis.

14 Creation of a voltage pulse cell LASER Detector = Photomultiplier tube pulse height Photomultiplier tubes collect photons of light and convert them to current. The Analog-to-Digital Converter assigns a digital value to the voltage pulse.

15 cell number Displaying flow cytometry data: 1 parameter Histogram size Forward Scatter

16 granularity SSC Side Scatter 2 parameters Dot Plot example: human peripheral blood leucocytes Granulocytes debris, dead cells Lymphocytes Forward Scatter size FSC Monocytes

17 Other cytograms Contour Plot Isometric Plot Greyscale Density Density Plot 3D Plot

18 Fluorescence Fluorescence / L A S E R absorption emission

19 Fluorochromes 1. Covalently bound to other probes: ANTIBODIES 2. Non covalently bound: fluorescent dyes which bind to DNA, dyes sensitive to ph, Ca(II)...

20 Count Side Scatter Propidium Iodide Cell Death Analysis: dead cells 11.7% living cells Forward Scatter Propidium iodide (PI) binds to DNA and it isn't cell permeant FITC living cells dead cells Propidium Iodide

21 Green Fluorescence Green Fluorescence Reactive Oxygen Species production: 2'7'-dichlorofluorescin (DCFH) ROS 2'7'-dichlorofluorescein (DCF) Green Fluorescence DCF CTRL MDMA Change in dichlorofluorescein fluorescence of mouse striatal synaptosomes after 1-h incubation at 37 C alone or with MDMA. (Chipana et al. Neuropharm 51 (4), 2006) Forward Scatter Forward Scatter

22 Side Scatter Reticulocyte Analysis: anaemia classification Human peripheral blood erytrocytes reticulocytes platelets reticulocytes + erytrocyte Forward Scatter RNA bind to RNA reticulocyte erytrocyte

23 Immunophenotyping: To evaluate individual cells for the presence and absence of specific antigens The Cluster of Differentiation is a protocol used for the identification of cell surface molecules present on LEUKOCYTES

24 Immunophenotyping: analysis of leukaemias and lymphomas submandibular gland biopsy B-cell lymphoid neoplasm A leukaemia or lymphoma will express a specific set of markers depending on which stage and pathway of blood cell differentiation are affected. CD5 + CD19 weak intensity FMC-7 + CD20 + kappa light chain + lambda light chain - Mantle cell lymphoma

25 Side Scatter Immunophenotyping: HIV infection leukocytes CD4+ lympohocyte CD4+lymphocytes 700 x 10 3 /ml HIV CD4+lymphocytes depletion CD4+ count to measure disease progression

26 Sorting: to capture and collect cells of interest for further analysis/use FACS = Fluorescence- Activated Cell Sorting

27 Sorting: to capture and collect cells of interest for further analysis/use FACS = Fluorescence- Activated Cell Sorting

28 Sorting: some clinical applications sperm sorting: sex selection (artificial insemination, in vitro fertilization) bone marrow transplant stem cells isolation

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31 DNA Analysis: G0 / M / Propidium Iodide is a red fluorescent molecule which binds to nucleic acids. information about cells ploidy distribution of cells across the cell cycle [Fine needle aspirate of a breast carcinoma] There are normal, diploid cells present together with an aneuploid TUMOUR.

32 tandem dyes Some fluorochromes used to label antibodies

33 SSC Gating Data from human peripheral blood leucocytes: FSC Lymphocytes

34 Features of the apoptotic cascade that can be observed using flow cytometry: Expression of proteins involved in apoptosis (immunofluorescence) Activation of caspases (rhodamine 110 attached to caspase-specific peptide) Changes in the mitochondrial membrane potential (TMRM, DiOC 6, rhodamine 123, JC-1) Changes in the plasma membrane (PI, 7-AAD, Annexin V) Cell shrinkage (Scatter Plot) Chromatin changes DNA degradation (PI, TUNEL assay)

35 An example of multiparametric analysis: Immature human thymocytes incubated with dexamethasone 7-AAD = 7-aminoactinomycin D

36 Others applications: Intracellular calcium ions Fluo-3, Fura Red Intracellular ph Carboxy SNARF -1 Intracellular glutathione Monobromobimane give a fluorescent conjugate with glutathione Drug uptake RNA content Acridine orange, Pyronin Y Cell proliferation 5 -bromodeoxyuridine (BrdUrd) is incorporated into the DNA in place of thymidine.

37 Side Scatter Cell Death Analysis: UV-B radiation-induced apoptosis in Jurkat cells Forward Scatter ANNEXIN V Propidium iodide (PI) marks cells with broken cell membrane Annexin V mark apoptotic cells

38 Creation of a voltage pulse LASER cell Photomultiplier tube Photomultiplier tubes collect photons of light and convert them to current. pulse height

39 The Analog-to-Digital Converter assigns a digital value to the voltage pulse LASER SSC FL1 FL2 FL3 FL4 Event # 1

40 DNA Analysis: karyotyping Hoechst binds to AT-rich regions chromomycin A3 binds to GC-rich regions chromomycin A3 A bivariate flow karyotype of a normal human female. Chromosome analysis and sorting

41 Flow Cytometric Analysis of Isolated Mitochondria (a) very small samples can be analyzed (5,000 10,000 mitochondria) (b) (c) preparations contaminated with other cellular constituents can be handled (using mitochondria-specific markers) potential identification of mitochondrial subpopulations (d) mitochondria can be sorted for further analysis of, e.g., protein profile. NAO = 10- nonyl acridine orange

42 Δψ m ROS

43 Mitochondria swelling induces a decrease in Side Scatter

44 Compensation

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52 Optics FL1 SSC 488/10 530/30 FL2 585/42 FL4 661/16 90/10 Beam Splitter DM 560SP Fluorescence / Side Scatter DM 640LP 670LP Half Mirror Fluorescence Collection Lens FL3 Blue Laser 488 nm Beam Combiner Red Laser 635 nm. Focusing Lens Flow Cell 488/10 FSC Forward Scatter (FACScalibur 4 colours) sample