Bi9393 Analytical cytometry Department of Cytokinetics Institute of Biophysics AVČR, vvi Královopolska 135 612 65 Brno e-mail: ksoucek @ ibp.cz phone: 541 517 166 Karel Souček, Ph.D. K. Souček Bi9393 Analytical cytometry What is the problem with multicolor detection? K. Souček Bi9393 Analytical cytometry Fluorescence signal compensation Spectral flow cytometry Conventional vs. spectral analysis Recomendation - Combine fluorochromes with appropriate brightness and low impact on resolution of other colors - Avoid fluorochromes that are challanging to combine - Assess the impact to biological resolution: bigger is not always better - Optimize your protocol: controls, controls, controls (shortcuts don't work) Applications of flow cytometry NUCLEIC ACID ANALYSIS cell cycle and ploidy DNA break analysis incorporation of BrDU cyclin expression DNA denaturation analysis CELL PHENOTYPE ANALYSIS immunophenotyping using CD antigens (detection of differentiation and tumor markers) detection of cytokine receptors CYTOGENETICS chromosome analysis STUDY OF CELLULAR FUNCTIONS viability determination of intracellular pH analysis of organelles and cytoskeleton determination of membrane potential oxidative flashover determination of intracellular Ca2+ determination of intracellular cytokines Natural Killer ligation of labelled cells analysis of reporter genes K. Souček Bi9393 Analytical cytometry Biological applications of flow cytometry ◼ proliferation analysis ◼ fluorescent proteins K. Souček Bi9393 Analytical cytometry Cell cycle d:\powerpoint\figures\chapter17\1708.jpg http://i.imgur.com/iq9cbSw.jpg What is important in sample preparation and marking... ◼ The sample preparation and labelling procedure cannot be generalised - it depends on the cell type and the specific analysis – suspension of single cells – vital signs – fixation (ethanol, formaldehyde) – permeabilization (detergents) – diffusion – active transport Cell cycle analysisCell cycle analysis • one of the oldest applications of flow cytometry, determination of the cell cycle phase by the amount of DNA • flow cytometry is a suitable method for rapid and accurate cell cycle determination • in a simple way, the DNA is stained with a fluorescent dye specific for DNA. • Propidium iodide 4′,6-diamidino-2-phenylindole (DAPI) - dramatically increase fluorescence upon binding to DNA. Permeabilization of the cytoplasmic membrane is required. • Hoechst 33342 • Vybrant® DyeCycle • DRAQ5 • Quaternary benzo[c]phenanthridine alkaloids (QBAs) I. Slaninova, J. Slanina and E. Taborska, "Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes," Cytometry A, vol. 71, no. 9, pp. 700-708, 2007. K. Souček Bi9393 Analytical cytometry G2 M G0 G1 s 0 200 400 600 800 1000 G0G1 s G2M DNA Analysis DNA content C o u n t 2N 4N Normal Cell CycleNormal Cell Cycle Purdue University Cytometry Laboratories - propidium iodide - DAPI - Hoechst 33342 - 7-AAD Cell cycle histogram analysis ◼ conventional analysis using histogram segments (regions) is not used ◼ it is necessary to use special software to model the distribution analysis of each phase Cell cycle histogram: gating strategy Debris Aggregates Dip G1 Dip G2 Dip S Detection of cells in a synchronized cell cycle File analyzed: SAMPLE2.FCS Date analysed: 16-Oct-2006 Model: 2DA0n_DSD_ASD Analysis type: Automatic analysis Diploid: 57.22 % Dip G1: 70.35 % at 75.05 Dip G2: 5.60 % at 150.10 Dip S: 24.05 % G2/G1: 2.00 %CV: 3.02 Aneuploid 1: 42.78 % An1 G1: 83.63 % at 100.15 An1 G2: 5.87 % at 200.30 An1 S: 10.50 % G2/G1: 2.00 %CV: 5.02 DI: 1.33 Total Aneuploid S-Phase: 10.50% Total S-Phase: 18.25 % Total B.A.D.: 11.22 % Debris: 19.13 % Aggregates: 3.96 % Modeled events: 31253 All cycle events: 24037 Cycle events per channel: 190 RCS: 0.842 Debris Aggregates Dip G1 Dip G2 Dip S An1 G1 An1 G2 An1 S Aneuploidy is an important diagnostic marker 450px-Native_American_tobacco_flower Analysis of ploidy in higher plants http://en.wikipedia.org/wiki/Image:Nativ e_American_tobacco_flower.jpg Alstroemeria caryophyllaceaNicotiana tabacum 400px-Corntassel_7095 http://en.wikipedia.org/wiki/I mage:Corntassel_7095.jpg Zea mays endosperm 28 days after pollination Cell cycle analysis- limitations Analysis of BrdU incorporation Analysis of BrdU incorporation K. Souček Bi9393 Analytical cytometry ◼ bromodeoxyuridine is incorporated into DNA instead of thymidine during S-phase ◼ after fixation and partial denaturation of DNA, BrdU can be detected using a specific fluorochromelabeled antibody ◼ in the last step we can stain the DNA Analysis of BrdU incorporation Analysis of BrdU incorporation File: HL60 K/24h Region % Gated R1 100.00 R2 35.48 R3 10.25 R4 47.87 R5 1.32 R4 R2 R3 R5 K. Souček Bi9393 Analytical cytometry Click azide/alkyne reaction Invitrogen Click-IT app (Invitrogen) Multiplex imaging with Click-iT® RNA assays. NIH3T3 cells were incubated with 1 mM EU, formaldehyde-fixed, and permeabilized with Triton® X-100. EU incorporated into newly synthesized RNA (red) in some cells was detected using the Click-iT® RNA Alexa Fluor® 594 Imaging Kit. Tubulin (green) was detected with anti-tubulin mouse IgG9 and visualized with Alexa Fluor® 488 goat anti-mouse IgG. Nuclei (blue) were stained with Hoechst 33342. Click-IT app (Invitrogen) DNA synthesis analysis (proliferation) Tritiated (3H)thymidine 3H-thymidine • Original method for measuring cell proliferation • Radioactive • Not compatible for multiplexed analyses 3H-thymidine BrdU (5-bromo-2'-deoxyuridine) BrdU Br Br Br Br Br Br Br Br BrdU Br Br Br Br BrdU Br Br Br Br BrdU Br Br Br Br BrdU • Non-radioactive • Multiplex compatible but, strand separation requirement for anti-BrdU access, can affect: • Ability for other antibodies to bind • Morphology • Ability for dyes that require dsDNA to bind efficiently, i.e., cell cycle dyes EdU (5-ethynyl-2'-deoxyuridine) Click-iT EdU Click-iT EdU Click-iT Edu • Non-radioactive • No DNA denaturation required • Simplified protocol • Small molecule detection • Multiplex compatible, including • Other antibodies • Dyes for cell cycle analysis DNA and RNA analysis Pyronin Y vs. Hoechst 33342 - Pyronin interacts with ds RNA and DNA but its binding to DNA is inhibited by the presence of Hoechst 33342 ◼ Acridine orange - emits red light when interacting with RNA and green light when interacting with DNA Detection of intracellular proteins in combination with DNA detection Detection of mitotic cells ◼ Histone H3 is specifically phosphorylated during mitosis (Ser10, Ser28, Thr11) ◼ DNA double labelling vs. H3-P identifies the cell population in M-phase Flow cytometry most common application s Viability assays (propidium iodide , Calcein AM , ...) Proliferation ( BrdU , EdU , mitosis - pH3) DNA damage (yH2AX,...) Cell Death analysis ( AnnexinV , Cleaved Caspase3, …) Cell Cycle (DNA content, Cell cycle modulation after treatment) Immunophenotype characterization of the cells (CSCs markers, differentiation, …) IMMUNOPHENOTYPING Principle: cells are stained with monoclonal antibodies conjugated to various fluorescent dyes and analyzed using flow cytometry Pros: simple, standard, broad spectrum of tested reagents, multiplexing Cons: not every epitope is fixable, compensation, possible artefacts from dying cells, dissociation of solid tissue may affect results Ermann , J. et al. (2015) Immune cell profiling to guide therapeutic decisions in rheumatic diseases Nat. Roar. Rheumatol . doi:10.1038/nrrheum.2015.71 VIABILITY using LIVE/DEAD fixable stains Principle: reaction of a fluorescent reactive dye with cellular amines, in necrotic cells react with free amines both in the interior and on the cell = intense staining, live cells stained on surface only = dim signal Pros: simple, wide spectrum of dyes, fixable, The ArC Amine Reactive Compensation Bead Kit Cons: live cells have signal, stain only in buffers w/o BSA or serum, Tris or azide Debris Aggregates Dip G1 Dip G2 Dip S Principle: DNA content measurement by fluorescent nucleic-acid-binding dyes Pros: simple, wide spectrum of dyes, in both native and fixed samples Cons: doublets > G2/M , single parameter≠ DNA synthesis, > CV if not fixed by organic solvents CELL CYCLE DNA SYNTHESIS using click azide /alkyne reaction Principle: direct measurement of DNA synthesis via visualization of incorporation of nucleoside analogue Pros: no DNA denaturation required, simplified protocol, small molecule detection, multiplex compatible Cons: high concentration of Cu in reaction = not compatible with all fluorochromes 5-Ethynyl-2'-deoxyuridine alkynes azide triazole DNA DAMAGE using γ H2A.X Principle: Phosphorylation of the Ser-139 residue of the histone variant H2A.X, forming γH2A.X, is an early cellular response to the induction of DNA doublestrand breaks Pros: in theory simple immuno-staining after fix&perm Cons: DSBs can also be intrinsic, occurring in healthy, untreated cells, DSBs are formed in the course of DNA fragmentation in apoptotic cells Huang X, Darzynkiewicz Z: Cytometric Assessment of Histone H2AX Phosphorylation. In DNA Repair Protocols: Mammalian Systems. Edited by Henderson DS. Totowa, NJ: Humana Press; 2006: 73-80 CTRL GEM APOPTOSIS detected via PARP cleavage or caspase-3 activation Principle: Cleaved Caspase-3 (Asp175) Antibody detects endogenous levels of the large fragment (17/19 kDa ) of activated caspase-3. Cleaved PARP (Asp214) detects endogenous levels of the large fragment (89 kDa ) PARP1 protein produced by caspase cleavage. Pros: simple immunostaining after fix&perm , validated antibodies available Cons: not every cell type or signal necessary activates cp-3 or leads to PARP cleavage, timing Untreated MG-132 DNA stain ◼ Violet laser DAPI, Hoechst 33342 FxCycle Violet, … ◼ Blue laser Selected Dyes , PI, … ◼ Red laser FxCycle Far Red 7-AAD Broad spectrum of the dyes Problem with: High concentration of dye, no wash Spillover & Compensations Compensation Antibody conjugates: • anti-rat and anti-hamster Igκ /negative control compensation beads (BD Biosciences), • Sphero TM Biotin Polystyrene Particles ( Spherotech , Lake Forest, IL, USA) Live/Dead fixable dyes: • ArcTM Amine Reactive Compensation Bead Kit beads ( Thermo Fisher Scientific) DNA stain: • fixed and permeabilized cells with/without appropriately diluted DNA probe Isotype controls were recorded for all samples. Gates were set according to isotype controls and control untreated cells (for γH2AX and cleaved caspase-3) Gating strategy included viability, discrimination of doublets (FSC-H vs. FSC-A) and debris (FSC vs. SSC). In samples with DNA marker, doublets we further discriminated using DNA marker (PO-PRO-1 A vs. PO-PRO-1 W). In the process of protocol optimization, FMO controls were measured and revealed DNA dye spillover. Detection of the number of cell divisions Fluorescent proteins ◼ bioluminescence resonance energy transfer (BRET) Aequorea victoria - a jellyfish that lives in the waters off the coast of North America. – is capable of blue luminescence (bioluminescence). Ca2+ interacts with the photoprotein aequorin. – blue light excites green fluorescent protein. Renilla reniformis - coral living in the waters off the north coast of Florida. – luminescence is produced by degradation of coelenterazine under the catalytic action of luciferase. – blue light excites green fluorescent protein. Renilla reniformis 'Sea Pansy' http://www.mbayaq.org/efc/living_species/default.asp?hOri=1&inhab=440 Aequorea victoria 'Crystal jelly' http://www.whitney.ufl.edu/species/seapansy.htm Fluorescent proteins ◼ Osamu Shimomura – 1961 discovered GFP and aequorin http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm Sci. 1994 Feb 11;263(5148): Green fluorescent protein as a marker for gene expression. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Department of Biological Sciences, Columbia University, New York, NY 10027. ◼ A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms. Fluorescent proteins ◼ Douglas Prasher ◼ Martin Chalfie Fluorescent proteins http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm in vivo molecular visualisation Hasegawa, S., Yang, M., Chishima, T., Miyagi, Y., Shimada, H., Moossa, A. R., and Hoffman, R. M. In vivo tumor delivery of the green fluorescent protein gene to report future occurrence of metastasis. Cancer Gene Ther, 7: 1336-1340, 2000. KODAK X-SIGHT 640 LSS Dyes in vivo with x-ray overlay KODAK X-SIGHT 650 and 761 Nanospheres, KODAK In-Vivo Multispectral Imaging System Fluorescent proteins ◼ Sergey A. Lukyanov – Discovers "GFP-like" proteins in nonluminous corals Roger Tsien ◼ ~ 2002 - mutation FP = colour spectrum http://www.tsienlab.ucsd.edu/ Debris Aggregates Dip G1 Dip G2 Dip S Detection of cells in a synchronized cell cycle Licensing control by Cdt1 and geminin J Cell Sci 2012 125: 2436-2445; doi: 10.1242/jcs.100883 Fucci (fluorescent ubiquitination-based cell cycle indicator) cells Ubiquitin E3 ligase complexes G1 - APCCdh1 substrate: Geminin, an inhibitor of DNA replication inhibits Cdt1 S, G2, M- SCFSkp2 substrate: DNA replication factorCdt1 - key licensing factor Fucci sensors - 1st generation, coral FP monomeric Kusabira orange 2 - hCdt1 (30/120) Monomeric Azami -Green – hGeminine (1/110) Fucci sensors - 2nd generation, Aequorea FP red monomeric fluorescent protein - mCherry -hCdt1 (30/120) yellowish green monomeric variant of GFP -mVenus – hGeminin (1/110) Fucci http://cfds.brain.riken.jp/Fucci.html CONTROL SCH900776 MU380VEHICLEGEMCITABINE ...lot of questions, but how to answer them? ◼ How many times cells divided? ◼ What is a length of cell cycle phases? ◼ Is there a difference in time between first and second division? ◼ How it is all affected by my drugs? Branches (dvisions) analysis 02_02_01_01 Median 14 hours 02_02_01_01 Summary of the lecture ◼ Compensation ◼ Quality control, principles ◼ proliferation analysis ◼ fluorescent proteins At the end of today's lecture, you should: 1. What are the basic principles of multispectral and mass cytometry 2. to know how the cell cycle can be analyzed. 3. be able to design another parameter that can be combined with DNA analysis. 4. know examples of cellular functions that can be analysed on a flow cytometer. 5. know what fluorescent proteins are and what are the advantages of their use in cell biology. 6. what click-IT is. At the end of today's lecture, you should: 1. What are the basic principles of multispectral and mass cytometry 2. to know how the cell cycle can be analyzed. 3. be able to design another parameter that can be combined with DNA analysis. 4. know examples of cellular functions that can be analysed on a flow cytometer. 5. know what fluorescent proteins are and what are the advantages of their use in cell biology. 6. what click-IT is. K. Souček Bi9393 Analytical cytometry Vital analysis of cellular functions ◼ Flow cytometry enables multi-colour vital analysis of cells – intracellular ion concentration, – pH, – production of reactive groups, – Lifetime Viability detection ◼ one of the simplest analyses ◼ works on the principle – detection of membrane integrity - impenetrability of some fluorescent markers through the cytoplasmic membrane of living cells - propidium iodide, ethidium bromide, 7-amino actinomycin D – detection of the physiological state of cells - use of fluorescent markers staining only living cells - Rhodamine-123, Calcein-AM ◼ ethidium monoazide - can be used to stain dead cells and then fixed ◼ With LDS-751 (laser dye styryl-751) it is possible to distinguish dead cells even after fixation ◼ LIVE/DEAD® Fixable Dead Cell Stain Kits Viability detection VIABILITY using LIVE/DEAD fixable stains Principle: reaction of a fluorescent reactive dye with cellular amines, in necrotic cells react with free amines both in the interior and on the cell = intense staining, live cells stained on surface only = dim signal Pros: simple, wide spectrum of dyes, fixable, The ArC Amine Reactive Compensation Bead Kit Cons: live cells have signal, stain only in buffers w/o BSA or serum, Tris or azide Signal transmission via Ca2+ •Cytosol (concentration - "resting" 100 nM vs. 1-10  activated) •[Ca2+ ]c activates protein kinase C •interacts with "Ca2+ - binding proteins" Alberts, B. et.al. Essential Cell Biology, 1998 K. Souček Bi9393 Analytical cytometry Ca2+ influx - Fura-2 - Fluo-3 - Indo-1 K. Souček Bi9393 Analytical cytometry ionophore ionophore Ensuring suitable conditions for detection [Ca2+ ]i •standardisation of staining and calibration - improved solubility of AM ester modified indicators (BSA, Pluronic ® -127) - inhibition of active secretion of the indicator by the cell (Probecid) - chelator modified AM esters (BAPTA-AM) suitable for calibration •Tempering of the sample throughout the measurement period •standardization of the inductor addition method K. Souček Bi9393 Analytical cytometry Burns, J. M. et.al. (1997). "Improved measurement of calcium mobilization by flow cytometry." Biotechniques 23(6): 1022-4, 1026. http://www.cytekdev.com K. Souček Bi9393 Analytical cytometry K. Souček Bi9393 Analytical cytometry Calibration (for one wavelength)   ( ) ( ) Ca K x F -F F -F 2 d min max + = R1 Fluo-3 (Kd ~ 400 nM, 22°C; 864 nM, 37°C) Fmin = 1.25 x FMnCl2 - 0.25 x Fmax K. Souček Bi9393 Analytical cytometry Intracellular pH detection ◼ Fluorescent markers that change fluorescence intensity as a function of pH ◼ SNARF-1, BCECF Intracellular pH detection ◼ Calibration with potassium buffers and ionophore (nigericin) required Detection of reactive oxygen species ◼ Reactive oxygen species play a key role in a wide range of biological processes – post-translational modification of proteins – transcription regulation – regulation of chromatin structure – signal transmission – immune system function – physical and metabolic stress – neurodegeneration, aging ABOUT2 - –O2 H2 O2 - OH H2 O 4 e– reduction to water e– e– e– e– Not so terribly reactive with most biomolecules Maintained at very low concentration Catalases, peroxidases, GSH, etc... Actually, a chemical reductant Not so terribly reactive with most biomolecules Mitochondrial superoxide the major source of active oxygen Maintained at very low concentration Superoxide dismutases Reacts with virtually any molecule at diffusion-limited rates The molecule that makes ionizing radiation toxic Unreactive at STP, but a great electron acceptor Biological activation via radicals, transition metals Generally, radical intermediates are enzyme- bound © Simon Melov Potential sites of intervention Oxidative Stress Neurodegeneration Cancer Vascular Tone Cardiac Output Sensory Acuity Skin Thickness Bone Density Endocrine Function Immune Function DNA Damage Oncogenesis Mitochondrial Damage Energy Crisis Cell Death © Simon Melov Hydroethidine HE EB N CH2 CH3 NH2H2 N H Br- N CH2 CH3 NH2H2 N + O2 Phagocytic Vacuole SOD H2 O2 NADPH NADP O2 NADPH Oxidase OH- O2 - DCF HE O2 H2 O2 DCF Example: Neutrophil Oxidative Burst © J. P. Robinson, Purdue University DCFH-DA DCFH DCF COOH H Cl O O-C-CH3 O CH3-C-O Cl O COOH H Cl OHHO Cl O COOH H Cl OHO Cl O Fluorescent Hydrolysis Oxidation 2',7'-dichlorofluorescin 2',7'-dichlorofluorescin diacetate 2',7'-dichlorofluorescein Cellular Esterases H2 O2 DCFH-DA DCFH-DA DCFH DCF H O2 2 Lymphocytes Monocytes Neutrophils log FITC Fluorescence . 1 1000100101 0 20 40 60 counts PMA-stimulated PMNControl 80 © J. P. Robinson, Purdue University Key Name K/72h+PMA ATRA/72h+PMA DMSO/72h+PMA NaBT/72h+PMA vit. D3/72h+PMA (A) (B) (C) - DCFH-DA - DHR-123 - HE Oxidative Burst K. Souček Bi9393 Analytical cytometry Fluorescent proteins ◼ bioluminescence resonance energy transfer (BRET) Aequorea victoria - a jellyfish that lives in the waters off the coast of North America. – is capable of blue luminescence (bioluminescence). Ca2+ interacts with the photoprotein aequorin. – blue light excites green fluorescent protein. Renilla reniformis - coral living in the waters off the north coast of Florida. – luminescence is produced by degradation of coelenterazine under the catalytic action of luciferase. – blue light excites green fluorescent protein. Renilla reniformis 'Sea Pansy' http://www.mbayaq.org/efc/living_species/default.asp?hOri=1&inhab=440 Aequorea victoria 'Crystal jelly' http://www.whitney.ufl.edu/species/seapansy.htm Fluorescent proteins http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm Fluorescent sensors for detection of H2 O2 Variants & fusions ◼ pHyPer-cyto vector ◼ pHyPer-dMito vector – Duplicated mitochondrial targeting sequence (MTS) is fused to the HyPer N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. ◼ pHyPer-nuc vector – Three copies of the nuclear localization signal (NLS) fused to the HyPer C-terminus provide for efficient translocation of HyPer to the nuclei of mammalian cells [Fischer-Fantuzzi and Vesco, 1988] Chromosome analysis and sorting Chromosome analysis and sorting ◼ synchronization of cells - gain of metaphase chromosomes (colcemid, hydroxyurea) ◼ chromosome isolation ◼ DAPI or Hoechst labelling vs. chromomycin A3 (CA3) or mithramycin = total DNA vs. G/C-rich regions NCBI PubChem logo http://www.scienceclarified.com/Ca-Ch/Chromosome.html http://www.nccr-oncology.ch/scripts/page9243.html Chromosome analysis and sorting e-bang "Flow karyotype" http://www.sanger.ac.uk/HGP/Cytogenetics/ Carcinoma of the bladder Sorting chromosomes Pisum sativum Sorting chromosomes Vicia faba Application of flow cytometry in microbiology ◼ Ecology ◼ food industry http://www.cyto.purdue.edu/flowcyt/research/micrflow/ Application of flow cytometry in microbiology Application of flow cytometry in microbiology ◼ viability ◼ metabolic functions ◼ sorting ◼ Aerosol analysis (Fluorescence Aerodynamic Particle Sizer (Flaps)) Application of flow cytometry in microbiology ◼ Sorting – EPICS + Autoclone® module Yeast flow cytometry ◼ cell division ◼ viability ◼ membrane potential ◼ respiration ◼ H production2 O2 ◼ sensitivity to antibiotics ◼ separation Saccharomyces cerevisiae http://en.wikipedia.org/wiki/Image:Budding_yeast_Lifecycle.png http://www.sbs.utexas.edu/mycology/sza_images_SEM.htm Yeast flow cytometry Flow cytometry in hydrobiology ◼ study of pico- and nanophytoplankton (< 20 ) ◼ analysis of plankton metabolic functions ◼ pigmentation study (chlorophyll and phycoerythrin analysis) Flow cytometry in hydrobiology Flow cytometry in hydrobiology ◼ DNA analysis http://planktonnet.awi.de/portal.php?pagetitle=assetfactsheet&asset_id=15127 http://www.soes.soton.ac.uk/staff/tt/ NCBI PubChem logo Flow cytometry in hydrobiology http://www.cyto.purdue.edu/flowcyt/research/micrflow/sieracki/sierack2.htm http://omlc.ogi.edu/spectra/PhotochemCAD/html/chl orophyll-a(MeOH).html Flow cytometry of invertebrates ◼ common methodological approaches and fluorescent markers can be applied ◼ Application examples: – cell cycle – Cytotoxicity – apoptosis Long Tongue Tachinid Fly 240px-Miesmuscheln_Mytilus_2 Invertebrate Survival Journal http://www.icms.qmul.ac.uk/flowcytometry/uses/insects/index.html http://www.icms.qmul.ac.uk/flowcytometry/uses/insects/index.html Figure 5. Representative flow-cytometry scatter plot of hemocytes from 25 oysters. Rebelo MdF, Figueiredo EdS, Mariante RM, Nóbrega A, et al. (2013) New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes. PLoS ONE 8(2): e57384. doi:10.1371/journal.pone.0057384 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057384 Figure 6. Proposed model for hemocyte maturation, as seen by flow cytometry. Rebelo MdF, Figueiredo EdS, Mariante RM, Nóbrega A, et al. (2013) New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes. PLoS ONE 8(2): e57384. doi:10.1371/journal.pone.0057384 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057384 https://www.union bio.com/copas/ Suraj Kannan. Circulation Research. Large Particle FluorescenceActivated Cell Sorting Enables High-Quality Single-Cell RNA Sequencing and Functional Analysis of Adult Cardiomyocytes, Volume: 125, Issue: 5, Pages: 567-569, DOI: (10.1161/CIRCRESAHA.119.315493) © 2019 American Heart Association, Inc. "High Throughput Flow Cytometry" ◼ automation + robotics = faster and more efficient data collection (measuring dozens of samples per hour with minimal operator intervention) ◼ using the principle of multicolour analysis K. Souček Bi9393 Analytical cytometry Automated sample measurement systems Automatic carousel (autosampler) Adapter for drawing samples from microtiter plates Automated "microsampler" system cytek K. Souček Bi9393 Analytical cytometry https://www.youtube.com/watch?v=L1UgdoP2aeg https://www.youtube.com/watch?v=7MFmbtIb8xA https://www.synbiobeta.com/read/one-lab-in-germany-is-using-robots- to-advance-computer-aided-synthetic-biology https://www.youtube.com/watch?v=ERDtmYddNkQ Incorporating Automation into a Flow Cytometry Workflow for Antibody Discovery The steps in a high-throughput fluorescence-microscopy experiment. Analysis http://www.stanford.edu/group/nolan/ http://www.stanford.edu/group/nolan/ Garry Nolan Peter Krutzik "Fluorescent cell barcoding" Krutzik PO, Nolan Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods. 2006 May;3(5):361-8. K. Souček Bi9393 Analytical cytometry Krutzik PO, Nolan Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods. 2006 May;3(5):361-8. K. Souček Bi9393 Analytical cytometry Get the best out of your model FACS-based surface screen: • validated antibodies in 96w plates • several comercially available possibilities, we have gone for... - LEGENDScreen HUMAN 332 PE conjugated antibodies + ISOs - LEGENDScreen MOUSE 252 PE conjugated antibodies + ISOs - there are XY vials in LN - price of kit ≈ 1000 € (27k Kc) How to get the best of it all? Final workflow The optimal concentration issue HOW TO TEST IT: 10x serial dilution REQUIREMENTS: ◼ optimal resolution ◼ compatibility w/ PE Sample results EpCAM - marker of epithelial cells - commonly lost during EMT Sample result Cytometric bead array (CBA) ◼ Multiplexed Bead-Based Immunoassays ◼ flow cytometry application that allows users to quantify multiple proteins simultaneously Multiplex microsphere-based flow cytometric platforms for protein analysis and their application in clinical proteomics - from assays to results ELECTROPHORESIS Volume 30, Issue 23, pages 4008-4019, 3 DEC 2009 DOI: 10.1002/elps.200900211 http://onlinelibrary.wiley.com/doi/10.1002/elps.200900211/full#fig1 CBA CBA ◼ multiplexing capabilities ◼ speed ◼ incorporation of multiple assay formats ◼ rapid assay development and reasonable cost ◼ automation ex vivo flow cytometry - limitations ◼ Influence of some cell properties (morphology, expression of traits); ◼ does not allow longer-term studies of cell metabolism and cellular interactions (communication, adhesion) in the natural tissue microenvironment; ◼ Next: – low sensitivity for detection of rare cell subpopulations (1-10 cells/ml ~ 5000 - 50000 cells in 5 litres of adult blood); – time-consuming sample preparation (hours, days); – discontinuity of sampling. Cytometry part A 79A: 737-745, 2011 in vivo flow cytometry - basic principles ◼ Imaging of cells directly in the blood or lymphatic system. ◼ Visualization using a CCD or CMOS camera after irradiation with a conventional microscope lamp or lasers. ◼ Detection of absorption, fluorescence, Raman spectra, photothermal or photoacoustic signals. Cytometry part A 79A: 737-745, 2011 in vivo flow cytometry Cytometry part A 79A: 737-745, 2011 in vivo flow cytometry - without labelling ◼ Record video using a high-speed, high-resolution CCD or CMOS camera in pass-through or bounce mode. ◼ Example: high-speed transmittance digital microscopy (TDM) ◼ Limits: tissue depth. ◼ TDM can be used to guide radiation sources to a designated area for further analysis. Cytometry part A 79A: 737-745, 2011 photoacoustic and photothermal imaging ◼ The photoacoustic effect was first discovered by Alexander Graham Bell in his search for a means of wireless communication.1 Bell succeeded in transmitting sound with an invention he called the "photophone," which carried a vocal signal with a beam of sunlight that was reflected by a vocally modulated mirror. The sound could be recovered with an ordinary telephone receiver connected to a selenium cell illuminated by the light. Bell published the results in a presentation to the American Association for the Advancement of Science in 1880. The Photoacoustic Effect Benjamin T. Spike Physics 325 April 21, 2006 Schematic illustration of photoacoustic imaging Cytometry part A 79A: 737-745, 2011 ACS Nano 2010 4 (8), 4559-4564 In vivo flow cytometry - detection of specific signals ◼ Detection of photoacoustic and photothermal phenomena Cytometry part A 79A: 737-745, 2011 in vivo flow cytometry - applications Summary of the lecture ◼ Examples of functional analyses ◼ "High-throughput" flow cytometry ... ◼ ... and the application of multicolour detection and beads array ◼ chromosome sorting ◼ applications in microbiology, hydrobiology and invertebrate studies ◼ in vivo flow cytometry At the end of today's lecture, you should: 1. to know how the cell cycle can be analyzed. 2. be able to design another parameter that can be combined with DNA analysis. 3. know examples of cellular functions that can be analysed on a flow cytometer. 4. know what fluorescent proteins are and what are the advantages of their use in cell biology. 5. know what "high-throughput" flow cytometry is ...and how the principle of multi-colour marking can be applied. 6. know the basic principles of measuring and sorting chromosomes using a flow cytometer; 7. have an idea of the possible applications of flow cytometry in microbiology, hydrobiology and invertebrate studies; 8. understand the limits and principles of in vivo flow cytometry. At the end of today's lecture, you should: 1. to know how the cell cycle can be analyzed. 2. be able to design another parameter that can be combined with DNA analysis. 3. know examples of cellular functions that can be analysed on a flow cytometer. 4. know what fluorescent proteins are and what are the advantages of their use in cell biology. 5. know what "high-throughput" flow cytometry is ...and how the principle of multi-colour marking can be applied. 6. know the basic principles of measuring and sorting chromosomes using a flow cytometer; 7. have an idea of the possible applications of flow cytometry in microbiology, hydrobiology and invertebrate studies; 8. understand the limits and principles of in vivo flow cytometry. K. Souček Bi9393 Analytical cytometry