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DVS User meeting (19.11.2013)

DVS User meeting (19.11.2013). DeCIPHer Initial Steps in Setting up a Mass Cytometry Experiment: Antibody Labelling and QC, Initial Gatings and some other Tips/Points Hervé LUCHE, PhD. Hosting and mastering all steps in a mouse project in immunology.

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DVS User meeting (19.11.2013)

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  1. DVS User meeting (19.11.2013) DeCIPHerInitial Steps in Setting up a Mass CytometryExperiment: Antibody Labelling and QC, Initial Gatings and some other Tips/Points Hervé LUCHE, PhD

  2. Hosting and mastering all steps in a mouse projectin immunology • 3500 m2 building dedicated to immunology • High end equipmentsin cytometry and imaging • One of the largestfacilitydedicated to immune challenges of mice in France • 40 highlyqualifiedtechnical staff to support youateverysteps of yourresearchproject Opening Jan 2014

  3. Standard high content MFC analysisalreadyavailable Thymus T CellDeveloppement 16 Parameters Spleen Orientation Panel 21 Parameters Lymphocyte-T 16 Parameters Lymphocyte-B 16 Parameters MyeloïdCells 19 Parameters WIP BoneMarrowPrecursors, BM B celldevelopment NK Cells

  4. Identification of a phenotype in a rare cell population using a 15 color, 17 Parameterpanel by conventional flow cytometry 25 cells in MUT versus 250 in CTRL out of 1E6 thymocytes

  5. Why do weneedthisguy for ?

  6. I have a dream… • Look atmanycell surface markers at once to preciselydefine the heterogeneity of an hematopoieticcompartment in one go • Look atthe signal transduction cascade in response to a modulation/stimulus • Look atmetabolic markers to definecell cycle status • Look atepigeneticlandmarks to followchromatinremodeling • Look at transcription factors for sub-lineagesspecification • In thesecells, look atintracellular cytokines production to precise the function of a cell IC antigendetectionrequires fixation conditions that are not nice to ourfluorochromes (especially tandems !)

  7. Panel design = Choose the best marker/fluorochromecombination • Antigen Classification to be used in a panel • Primary: • Well characterized, easily classified positive or negative (CD3, CD4, CD8) • 2. Secondary: • Well characterized, also expressed at a higher density, often over a continuum (CD24, CD11b, etc…) • 3. Tertiary: • Expressed only at low levels (CD25), also uncharacterized antigens –often available in only one or two colors • 4. Quaternary : • Well characterized not mandatory but increase coherence and robustness Not an easytask to establish panels >10 parameters… Mahnke, Y, Clin Lab Med, 37(2):364-76, Au 2007:” Optimizing a Multi-colourImmunophenotyping Assay.”

  8. The wayweproceeded to get mass cytometry running at CIPHE Generate data to finalise a paper on thymocyte development Design yourexperimentalproceduresgradually to understandwhatyou are doing !

  9. How to setup a CyTOF experiment ? • Configure your instrument (laser power, BP/LP/ND filters) • Characterize your instrument (CSTs and QCs) • Design your panel accordingly • Optimize settings for your assay (2,5 SDEN, Lin Max) • Run appropriate controls (Comp Beads, FMOs for continuums) • QC your data (Dead cells, Doublets, Clogs, Mis-annotations) ! • Use adapted analysis tools • Configure your instrument (QC, Mass Range, Dual Calibration) • Characterize your instrument (Sensitivity across Mass range) • Optimize settings for your assay (Nebular Flow for Oxydation) • Design your panel accordingly • Run appropriate controls (Eu/eQ/Comp Beads, Cells + DNA) • QC your data (Dead cells, Ion Cloud Fusion, Doublets) • Use adapted analysis tools (Cytobank + SPADE, VisNE)

  10. Sensitivity variation across the mass range • The stain index in mass cytometry is less than conventionnal fluorescent dyes • All metal are not equivalent in term of sensitivity • Know your instrument ! • Use metalelements of the maxPar kit and runthem on the CyTOF (H. MAEKER) • Use trace multi-element solution (A. COSMA)

  11. Is this difference in sensitivity really significant ? • Test sensitivity issues: • Label 3 times CD45.1 and CD45.2 withmetalatextremities of the mass range + the middle part. • Mix splenocytesfrombothCD45.1 vs CD45.2 congenic C57BL/6 mouse strainsat3 different ratios: • 3/97, 50/50, 97/3 • Stain the cellswith the appropriatecombination of antibody(1 ugeach) Is the difference in stainingintensityobserved in eachcombinationdetrimental for an accuratedetermination of cellfrequency?

  12. The difference in sensitivity is really minimal 1µg of CD45.2-141Nd CD45.2-141Nd-2013JL26 61 atoms/mAb Intensity signal : 63 1µg of CD45.2-156Gd CD45.2-156Gd-2013SE03 61 atoms/mAb Intensity signal : 342 1µg of CD45.2-176Yb CD45.2-176Yb-2013JL05 47 atoms/mAb Intensity signal : 207 Same trend observedfittingwithintrumentsensitivitycurves but this has no impact on the frequenciesobserved. Even in the worstcombination, wecanaccuratelymeasure the cellfrequency of eachgenotype Experiment: 13-SE19-TEST-MMK

  13. The difference in sensitivity is really minimal The normalised intensity of the staining follow the same trend as the one observed with the multi-element solution

  14. Some degree of spillover observed (-1, +1 and +16) but not requiring compensation CyTOF Channel Single-Isotope Sample There can still be signal interference/overlap from isotopic impurities in the metal reporters (usually + and/or – 1 Da) and oxidation (+16 Da) of the reporter ions during analysis With the kindpermission of H Maeker

  15. Building our metal-labeled antibody biobank

  16. Antibody labeling and QC Procedure • Check the frequency of targetcell population in the panel • To alleviate the potentialpitfalls of alteredantigen recognition uponMaxParconjugation, detect the targetfrequencyusingprimaryantibodiescoupled to fluorescent dye or biotin • Do a secondarystainingusinganti-PE, FITC, APC or biotincoupled to metals (DVS) • Label yourantibody: • Do the labeling • Determine the final antibody concentration using the nanodrop • Measure the amount of elementscoupled to antibody by incubating and running itintoacid. • Use Batlab to calculate the number of atombound per antibody • QC Tips • Use OneCompBead (eBioscience) to quickly test for antibodylabeling to metal

  17. Determining the number of atoms bound per antibody using Batlab

  18. Example of antibody labeling Flow (mAb-PE) CyTOF (anti-PE) CD172a CyTOF DCs Specificity CD11b- on spleen; CD172a-PE BD Clone: P84 1/100 Up: on Dentriticcells (CD5- CMHII+ CD11c) Down: on Tcells pop (CD5+ CMHII-) Exp: 13-JL08-TEST-MMK FLOW Illustration: CD172a+ histogram CD172-167Er BD Clone: LG.3A10 692µg/ml / 57 atoms/mAb 1/100 CD172a-PE I) BD Clone: P84 1/100 II) DVS anti-PE-156 1/100 Atthis time, DVS doesn’tsell a conjugated CD27 mAb.

  19. Likewithother class of cytometers, itisreallyeasy to generate artefacts on a CyTOF ! B cells MHCII CD19 T cells CD5 CD8 CD4 Ion cloud fusion events or cell doublets could explain these staining artefacts !

  20. Try to keepyourcellsalong the whole of yourprocedure ! • Basic parameter to tune in order to get this problem fixed: • Determine the optimal cell concentration of the sample on your machine CellLength Single Event Ir191 Doublet Ir193 (x1E6 cells/mL) In our operating conditions, we have an optimal ratio of singlets/doublets for a cell concentration of 0.5x1E6 cells/mL Ir191

  21. How to increasethe efficacity of single events for hypocellularsamples ? • Spike your sample with cells of irrelevant background (here 1xE6 Jurkat cells) • Can be useful to determine your background in every channels

  22. Change of procedure: runstainedsamplesnextday Cells are lost in somestoring conditions ! It seems a short fixation is not enough for a good conservation of the samples How to store adequatelysamples to runthem the nextday ? Cellnumber In our hands, a short fixation withCytoFIXfollowed by conservation overnight in CytoFIX gives the best recovery of cells (30% of input cells) The residualcelllosswas due to the use of PS tubes for the step in H20 and not PP tubes !

  23. Try out Home Made Barecodingusing CD45.2 antibodiescoupled to 4 differentmetals • Simple goals: • Reduce the time of acquisition/washing of each dilution serie of a mAb titration • See the principle of Barecoding + amendability • Experimental design: • Use anti-CD45 antibodies self labeled to Sm147, Pr141, Gd156, Lu176 usingMaxPar Kits • Each of thesewereused to code for one giventiterserie of the titration range • Tips • Use InfinicytMultiDimensionalCytogram • to debarcode • Not as complex as dyebarcoding: • Wait for DVS Science Commercial Kit !

  24. Why do one need to normalise a dataset or a file resultingfrom a long acquisition ? • Takeadvantage of the home-made barcodingexperiment • 6000 seconds acquisition (1h 45min around) • Look at the evolution of the signal for severalmetals • Cut the time space in 4 equal slices % IntensityLoss over time Signal Intensity Drift over time As an average for all channelsanalysed in thisexperiment, the signal intensitywasdecreased by 12% at the end of acquisition Use normalisation beads to getrid off this drift and compare datasets

  25. Data normalisation usingeQbeads • EQ Beadsfrom DVS (#201078) • - 4 elementsmetal-embeddedpolystyrenebeads for data normalisation • - 7 Isotopes (140Ce, 142Ce, 175Lu, 176Lu, 165Ho, 151Eu and 153Eu) • Procedure • Download Normalisation software • Tips • Select all isotopes to limit visualisation artefacts

  26. Currentgatingmethodis an issue if youwant to look at quiescent cells AND proliferatingcells Activated T cells Splenocytes « Singlets » gatepasted on proliferating T cells Cells in Proliferation non-prolifcells « Proliferation » gatereported on non-proliferatingcells (Conc > 5.10E05 cells/ml on purpose to generate visible doublets/ion cloud fusion) 65 If the initial gatingisbased on the removal of DNA high cells, half of the proliferatingcells are lost !

  27. Why Ki67 is not the best marker to look atproliferatingcells

  28. Use a combination of CD71 and IdU to mapproliferatingcells : • Use a combination of CD71 and IdU to mapproliferatingcells IdUi.p. (+1h) IdU+ cells IdUi.p. (o/n) IdU+ cells No IdU IdUisreallyeasy to apply in contrast to BrdUanalysis by conventional flow !

  29. 16 anti-mouse mAb MAXPAR® Panel Kit Panel Catalog N° #201306 Package Size 25 tests Kind Gift from DVS Science for this training ! • 17 surface markers • 1 nuclearantigen • 1 BrdUanalog • Live/Dead DNA staining • = 22 parametersmeasured

  30. Experimental design for the workshop Modulation Group 1 Group 2 • Compare flow and mass cytometryresultswithsimilarmAb panels • Datasetfreelyavailable on INSERM Cytobank on demand

  31. Mix Storage over time: not a good idea if pre-mix isdiluted

  32. Effect of inappropriate initial fixation for intranuclearstaining

  33. Additional tests we have performedat CIPHE • Antibodieslabeled to colloidal gold (normallyused for ElectronMicroscopy) • Streptavidin Au197-A488 10 nm colloidal gold conjugate (Invitrogen Cat A32361) • QdotsfromInvitrogen : • Nanocristals of cadmium mixed with selenium or tellurium • coated with an additional semiconductor shell (zinc sulfide) • CD114 (28%) is a good channel • The bigger the better ? (good signal with Qdot655) • Primaryor secondarystaining • Qdots are not stable ! Use not olderthan 6 months ! ❷ Anti-fluorochrome (PE, FITC, APC) ❸Anti-biotine (differentmetals) . veryusefulto test a new mAb in your panel . Wetried to conjugatestreptavidine (DOTA maleimideactivated); lowefficiency

  34. Other collaborators DVS Science ! Cytobank Stephane MANCINI, PhD (CIML) Jacques NUNES, PhD (CRCM) Antonio COSMA, PhD (CEA) Joel SEDERSTROM, IE (BaylorCollege) Thure ADLER, PhD (German Mouse Clinic) Ryan BRINKMAN, PhD (BCCA) Mike ROONEY Jane LIMER, PhD Alex DARMOISE, PhD NikeshKotecha, PhD Tiffany CHEN, PhD Centre d’Immuno-phenotypage (CIPHE) Bernard & Marie MALISSEN Lab (CIML) Marie MALISSEN, PhD, Head of Immunophenotyping Module Mickael MEYRAND, CyTOF Specialist Pierre GRENOT, Cell Sorting Specialist Marielle MELLO, Flow Cytometry and Bioassays Specialist Lillia HADJEM, Flow Cytometry and Bioassays Specialis Ana ZARUBICA, PhD, Chief Operating Officer Romain RONCAGALLI, PhD Laurence ARDOUIN-BATAILLE, PhD Claude GREGOIRE, PhD Sandrine HENRI, PhD Dora THERHORST, MD Samira TAMOUTOUNOUR, PhD student

  35. Your contact @ CIPHE Dr Bernard Malissen Head of the Institute bernardm@ciml.univ-mrs.fr Dr Ana Zarubica Chief Operating Officer ana.zarubica@inserm.fr Dr FredericFiore, Head of GEM generation frederic.fiore@inserm.fr Dr Marie Malissen, Head of Immunophenotyping and Cryoconservation Module marie.malissen@inserm.fr Dr Jean-Pierre Gorvel Manager BSL3-Bioimaging gorvel@ciml.univ-mrs.fr Philippe Hoest BiosecurityOfficer BSL3-Bioimaging philippe.hoest@inserm.fr Anne Gillet Head of Animal House anne.gillet@inserm.fr Dr Herve Luche R&D Manager Immunophenotyping Module herve.luche@inserm.fr

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