Flow Cytometry and Sorting

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1 Flow Cytometry and Sorting Much of this information was gleaned from the internet from sites such as Purdue University Cytometry Laboratories, The Scripps Research Institute, and BD Biosciences. Barbara Walter UCR IIGB 2023 Keen Hall

2 What is Flow Cytometry? "Cytometry refers to the measurement of physical and /or chemical characteristics of cells, or, by extension, of other biological particles. Flow Cytometry is a process in which such measurements are made while the cells or particles pass, preferably in single file, through the measuring apparatus in a fluid stream. Flow Sorting extends flow cytometry by using electrical or mechanical means to divert and collect cells with one or more measured characteristics falling within a range or ranges of values set by the user." The term "FACS" is Becton-Dickinson's registered trademark and is an acronym for Fluorescence-Activated Cell Sorter. This definition for Flow Cytometry is adapted from the third edition of: Howard Shapiro: "Practical Flow Cytometry". John Wiley and Sons, Inc., New York.

3 A simplified illustration of Flow Cytometry

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6 Compensation Each fluorochrome has its own excitation spectrum (the range of illuminating light wavelengths that will cause it to fluoresce) and an emission spectrum (the spectrum of fluorescent light emitted). Since flow cytometers and confocal microscopes use laser light for illumination, the laser wavelength must be within the excitation spectrum. To use two dyes simultaneously, both of their excitation spectra must overlap the laser wavelength but their emission spectra must differ. More flexibility of choice is afforded where more than one laser is used.

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9 The power of cytometry arises from our ability to simultaneously mark and analyze several different cellular structures or properties with fluorescent dyes. Immunofluorescence: In the immunofluorescence technique, fluorescent dye is carried to the cell by antibody molecules (usually monoclonal) that are specific for some cellular receptor. There are a wide range of fluorochromes that may be directly conjugated to antibody molecules or obtained already attached to forms of avidin, ready for subsequent binding to biotinilated antibody. Common examples are fluorescein, phycoerythrin (PE), allophycocyanine (APC), texas red, the cyanine dyes Cy3, Cy5 and Cy7 and Molecular Probes' Alexa dyes. The Dyes That Bind: There are dyes that bind of their own accord specifically to intra-cellular sites. for example, Propidium Iodide binds to nucleic acid (DNA or RNA), Hoechst binds to AT-rich regions of double-stranded DNA, Rhodamine123 binds to mitochondria. Keeping Track: Tracking dyes (e.g. CFSE, PKH26) bind tenaciously to cells, surviving for days in vivo, and even through several cell divisions, upon each of which the fluorochrome is divided between the daughter cells. Intra-Cellular Chemistry: Using time as a parameter is useful. Some fluorochromes are even sensitive to the local chemistry (Fluo-3 responds to intra-cellular Ca++ concentration, BCECF to intracellular ph).

10 CELLULAR ANTIGENS cytokines structure enzymes Adhesion Metabolic Receptors courtesy of Jim Bender

11 Schematic diagram of the basic unit of immunoglobulin (antibody) 1. Fab 2. Fc 3. heavy chain (consist of VH, CH1, hinge, CH2 and CH3 regions: from N- term) 4. light chain (consist of VL and CL regions: from N-term) 5. antigen binding site 6. hinge regions (*) -S-S- mean disulfide bonds.

12 WAYS ANTIBODIES BIND TO CELLS Specific: Fab to epitope Fc to Fc receptor binding is high affinity and saturable Non Specific: binding is low affinity and not saturable

13 INDIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING Primary Antibody: murine monoclonal antibody Fc Fab Second Antibody: fluoresceinated goat anti-mouse IgG F(ab') 2 FcR A B C D epitope

14 BLOCKING WITH GOAT IgG goat IgG

15 ISOTYPE CONTROL- myeloma protein E F G H AUTOFLUORESCENCE CONTROL

16 AMOUNT BOUND SPECIFIC ANTIBODY NON-SPECIFIC ANTIBODY CONCENTRATION

17 Uncompensated Dot Blot Compensated Dot Blot Contour Plot Histogram Histogram of gated P6 and not P6

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24 Cell Tracking Using CFSE Cell divisions Number of events Log Scale of CFSE Fluorescence CellTrace CFSE Cell Proliferation Kit - for flow cytometry Catalog Number - C34554 Molecular Probes

25 Analysis Software FlowJo; MAC in main lab ModFit LT; MAC in main lab ; CellQuestPro on FACScan computer FACS Diva on FACSAria computer

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31 Cell Preparation Consideration for Sorting Only happy cells will be successfully sorted The process of sorting is inherently stressful to cells and this must be considered in experimental design Proper buffers are essential to good sorts and there is no single recipe for all cells

32 Before After FACSAria High Speed Cell Sorting sample: 50 million cells flow: 6000 events/s yield: 3 million GFP+ cells Affymetrix GeneChip Array RNA DNA

33 Services available on the FACSAria Single or multi-color cellular analysis (phenotyping, intracellular staining, etc.) Sorting up to four different subsets at a time Sorting into plates single cell up to any stated number of cells sorted into a variety of plates (6-well, 96-well, etc.)

34 Uses for Flow cytometry DNA content Cell Cycle Apoptosis Telomere length RNA content Surface and intracellular antigens Ligand binding Surface charge Membrane integrity Membrane fusion/turnover Membrane organization Membrane fluidity Membrane permeability (dye/drug uptake/efflux) Endocytosis Cell Division/Generation number Enzyme activity Cytoplasmic Membrane potential Intracellular ph Gene expression Measure Cell activation Calcium levels

35 Helpful Training Links _systems/support/training/online/ rt/tutorials.html ensation-howto.html r/tools/index.jsp

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