FACS Lecture Feb 2020 PDF

FACS Fluorescent Activated Cell Sorting Facial Acting Coding System Factors Affecting Cesarean Section Dr. Ruth Beckervordersandforth Institut für Biochemie  Flow cytometry Analysis of physical and chemical properties...

FACS Fluorescent Activated Cell Sorting Facial Acting Coding System Factors Affecting Cesarean Section Dr. Ruth Beckervordersandforth Institut für Biochemie  Flow cytometry Analysis of physical and chemical properties of single particles (cells) passing voltage or light source with high speed. Allows for separation of single particle (cells) from a heterogeneous population. Fluorescent Activated Cell Sorting based on fluorescent-labeles cells  Other separationforms for cell populations cell filtration cell affinity methods fractioning elutriation MACS = Magnetic Activated Cell Sorting - FACS is very versatile (optics) - analysis and sorting of up to 4 populations at the same time - FACS machines are very expensive - need specifically trained user  What to analyse and sort for? mammalian cells, yeast, bacteria, plant cells subcellular organells: Goldgi complex, chromosomes, sperm For what? analysis sorting size/volume/complexity cell culture cell cycle functional assays RNA-/DNA-content transplantation protein expression enzymatic activity microarray-analysis apoptosis single cell-PCR MDR in cancer cells cloning of single particles chromosomes: human Genome Sequencing Project sperms: sex selection for in vitro fertilisation hematology, tumor-Immunology, chemotherapy, prenatal diagnostics Which parameters are messured with FACS? blood cells chromosomes algae protozoa pictures modified from BD  Which parameters are messured with FACS? basophile granulocytes T-cells monocytes erythrocytes neutrophile eosinophile granulocytes granulocytes cell are morphologically distinct based on size granularity  cellular parameters: relative size and complexity coherent light source forward scatter (488nm) size (488nm) side scatter granularity (488nm) Forward scatter (FSC) Side scatter (SSC) measured along the axis of incoming measured in 90° direction to exitation light light propotional to the cell size / cell proportional to cell „complexity“ or surface (only true for perfectly round granularity cell)  cellular parameters: example for light scattering in whole blood Side scatter granulocytes monocytes debris lymphocytes Forward scatter  cellular parameters: fluorochrome detection and quantification The fluorochrome molecule absorbes energy of the incoming light. It releases the absorbed energy by vibration and dissipated heat emission of a photon of higher wavelenght ( = less energetic).  cellular parameters: fluorochrome detection and quantification Fluorescent signals are proportional to numbers of fluorochromes bound to cells. FACS optical system fluidics electronics optical system  optical system: lasers Exitation of fluorchromes by different lasers  optical system: lasers Exitation of fluorescent dyes by different lasers  optical system Lasers create photons light output is - monochromatic and in phasen - unidirectional functions Detectors Filters/dichroid mirrors converts photons into electrons photon-distribution to detectors according to energy-levels (wavelengths)  optical system Lasers create photons light output is - monochromatic and in phasen - unidirectional functions Detectors Filters/dichroid mirrors converts photons into electrons photon-distribution to detectors according to energy-levels (wavelengths)  optical system: Filters Distribution of photons to detectors is filter-dependend.  optical system Lasers creates photons light output is - monochromatic and in phasen - unidirectional functions Detectors Filters/dichroid mirrors converts photons into electrons photon-distribution to detectors according to energy-levels (wavelengths)  optical system: Detectors Photons (scattered from cells or emitted from fluorochromes) have to be converted into electrons (electrical signal) to become analysed. Photodiodes: - detected parameter: FCS - direct and proportional 1:1 conversion of photons into electrons - no amplification inside the photodiode Photomultiplier tube (PMT) - detects FCS and fluorochromes - efficiency of photon to electron conversion is wavelengths-dependent - amplification inside PMT via dynodes Fluidics  Fluidics – hydrodynamic focussing cuvette: hydrodynamic focussing align cells while passing the interogation point for analysis sheath pressure: drives sheath buffer through cuvette sample pressure: higher than sheath pressure; delivers sample to cuvette; determines the flow rate general principle: drop formation and charging sample reaches cuvette passes nozzle vibration separates stream into single droplets droplets pass laser (analysis) droplets are electrically charged droplets pass deflection plates collection in collection tubes or pass to waste Fluidics – dropformation and charging  Electronics How to collect and display data? cell passes lasers photon send to detectors photon converted into electron electron converted into numerical value event intensity depicted as numerical value  Electronics How to collect and display data?  Electronics How to graphically depict data? histogramm dotplot Both plots can be generated linear or logarithmic. Biexponential transition from logscale to linear.  Electronics How to graphically depict data? dotplots 2dimensional representation of correlation of 2 parameters at least for posibilities single positive double positive double negative  examples dotplots WT TG APOPTOTIC CELLS Ist LIVING CELLS gate DEAD CELLS FSC-W BIGGER FITC SSC CELLS FSC FSC-A FSC-H 1. gate: dead cells single positive PE double positive population population (FSC - SSC) 2. gate: cell duplets (FSC height - FCS width) 3. gate: fluorophore 1 (FSC - FITC) 4. gate: double-labelled cells (PE - FITC) PE FITC negative single positive FITC  Sorting – general principles Stream stability is depedend on precise adaption between sheath pressure frequency nozzle size amplitude  Sorting – general principles frequency (number of droplets per second) determines the speed of sorting nozzle size and sheath pressure influence cell survival - nozzle size divided by 3 = biggest sorted cell - the more active a cell (division, phagocytotic activity, production of cytokines...), the more fragile. Conditions need to be adopted for each cell type!  Sort setup How to design a FACS experiment? Which cells should be analysed and what do I need to know to do so? cell size - which nozzle to use? cell preparation different preparation methods for: - cell suspension (e.g. blood, suspension cell culture) - adherent cells (e.g. cell culture) - cells from solid tissue (e.g. brain) The success of a FACSort is almost exclusively dependent on the sample condition. Cells or particles need to be sufficiently separated!!!  Sort setup How to design a FACS experiment? What do I want to use the sorted cells for? cell culture funktional assays transplantation microarray-analyse single cell-PCR cloning This determines - how many cells need to be sorted - how to collect the cells - how sterile I need to work  Sort setup How to design a FACS experiment? Which parameters do I want to analyse and what do I need for that? antigen expression fluorescent protein expression nucleic acid content functionality Choice of antibodies/fluorescent dyes/fluorochromes? Which parameters need to be considered for FACS? Compensation, negative controls and optimal dyes  Compensation Correction of spectral overlap between fluorophores Why and when? emission 480 520 560 600 640 680 720 wavelenght (nm)  Compensation 530/30 585/42 filter filter alexa 488 PE emission PE overlap alexa 488 overlap 480 520 560 600 640 680 720 wavelenght (nm)  Compensation 530/30 585/42 filter filter alexa 488 emission FL1 FL2 480 520 560 600 640 680 720 wavelenght (nm)  without compensation 105 104 PE fluorescence 103 102 0 102 102 0 102 103 104 105 alexa 488 fluorescence  compensation 105 104 PE fluorescence 103 102 0 102 102 0 102 103 104 105 a alexa 488 fluorescence  Gating und negative controls Problem: discriminating between specific staining and unspecific background  negative controls only secondary antibodies primary and secondary antibodies  Gating und negative controls 3d MCSFR-/- differentiation transduced time lapse progenitors GMPs M-CSF gated on live cells, singlets, MacI-, FcyR+ Bl6 (MCSFR+/+) unstained MCSFR-/- FSC-H MCSFR antibody does not have detrimental effect on sorted cells rescue of MCSFR-/- time lapse M-CSF MCSFR-PE courtesy of Max Endele  negative controls fluorescent-coupled antibodies Isotype controls - same species - same isotype-subtype - coupled to the same fluorophore - same concentration - same antibody-/fluorochrome-ratio fluorescent dyes unstained samples fluorescent proteins (transgenic mice) siblings that do not carry the fluorescent protein wildtype animals (same age, same genetic background) fluorescent proteins (transfected cells) control transfections (for example with plasmids that do not code for fluorescent proteins)  optimal choice of fluorochromes Multicolour experimente Brightness of fluorochrom need to match expression levels of antigen.  FACS experiment – process and result Fischer, Beckervordersandforth et al., 2011 Nature Protocols  FACS experiment – process and result Fischer, Beckervordersandforth et al., 2011 Nature Protocols  Sort setup How to design a FACS experiment Limiting factors FACS set up - laser/filter - spektral overlap limits number of fluorescent dyes Fluorescent proteins - cell surface proteins - not all antibodies work in FACS (best fluorophore-coupled antibodies)  latest developments in flow cytometry Improvements and extensions intracellular flow cytometry (Phospho-FACS) Quantum-Dot technology single cell masscytometrie Image activated cell sorting (IACS)  Intracellular flow cytometry Phospho-FACS for analysis of intracellular phosphorylation-dependent signaling pathways on single cell level diagnostic tests statistics of signaling cascades drug-screening  Intracellular flow cytometry Phospho-FACS staining with antibodies against cell surface proteins fixation (dependent on cell type) - 10-15 minutes in 2%PFA, - 5-10 minutes in 95%MeOH or Saponin staining with primary antibodies conjugated with phospho- epitopes (in PBS and 1%BSA)  Quantum Dot technologie Quantum Dots replace fluorophores consists of crystalline semi-conductors (Kadmium, Selenium; 2-6nm big) nano-crystals emit light of distinct wavelenghts in a size-depending fashion Advantages exitable by any laser 90% of the absorbed energy will be emitted as light; much better resolution of weak markers emissionsspectra are symmetric and show less overlap Disadvantages Difficult to produce few antibody exist until now not applicable with buffers containing e.g. heavy metal Well suited for multicolour experiments. Not feasible today but seminal.  Index sorting FACS issues: FSC and SCS à only general cell properties Fluorescent-based sorting à need to be carefully established in regard to permeablisation, labeling à requires a priori knowledge about cell-specific markers àavalability of commercial antibodies/transgenic constructs à low purity / yield with current protocols for certain cell types Gate placement manually à subjective decision Index sorting allows the isolation of single cells with retrospective analysis of each single cells‘ high dimensional immune phenotype. retrospective analysis by Mass spectometry and/or single cell sequencing  Single-Cell Mass Cytometrie Mass cytometry - antibodies are coupled to isotopes of transition elements - antibodies bind to specific epitopes in and at the cells - cells bound to antibody-isotope-conjugate are vaporized - induces ionisation of atomic components - ions are quantified via TOF-spectrometry Single cell level up to 100 parameters can be theoretically analysed (in practise about 40 parameters) can be combined with FACS and Phosphos-FACS Ultimate possibility to define and analyse signal responses in single cell populations as well as crosstalk between several defined cell populations. Single cell analyses provides overall view of e.g. immunresponse in case of disease or medical tests.  Index sorting Image activated cell sorting (IACS) Cell type purification by SC Transcriptome trained Sorting unbiased cell type identification by scRNA seq + FACS (high throuput measurements of cellular properties + Gate ID (computantional algorithm based on scRNA-seq data to predict optimal gating (Baron et al., 2019, Cell)  Index sorting Image activated cell sorting (IACS) Cell type-specific enrichment without antibodies/transgenes Simultaneous purification of many cell types (dependent on FACS space distinction) Trainings data sets are reusable and can be used to implement maschine- learning for automated cell type-purification as a routine FACS tool high costs (due to sequencing) cell types overlap in FACS space and cannot be separated properly longitudinal processes difficult to monitor (development and differentiated tissue à new cells arise) (Baron et al., 2019, Cell) Thanks Beckervordersandforth, Ruth Group leader, Institute of Biochemistry Telefon: (09131) 85-26206 [email protected]  Wahlfachstudenten 16.1.2017 Please be at 10:00 at the OICE (optical imaging centre Erlangen) Hartmannstraße 14, 91052 Erlangen www.oice.uni-erlangen.de Dr. Ralf Palmisano will welcome you there  Lasers, filters and dyes Cascade 440/40 Blue Krypton 540/80 Laser Alexa 430 407 nm 525/50 Fluorescein PE 575/25 12-color FACS Spectra of dyes 630/22 TRPE Argon Laser 665/30 488 nm Cy5PE 720/45 Cy5.5PE 785/50 Cy7PE 625/40 Alexa 594 Excitation spectrum 660/40 APC Dye Laser Emission 705/50 595 nm spectrum Cy5.5APC Collection Filter 750LP (wavelength/bandpass) Cy7APC 300 400 500 600 700 800 Wavelength (nm)