Bi9393 Analytická cytometrie Oddělení cytokinetiky Biofyzikální ústav AVČR, v.v.i. Královopolská 135 612 65 Brno e-mail: ksoucek@ibp.cz tel.: 541 517 166 •Karel Souček, Ph.D. •K. Souček Bi9393 Analytická cytometrie Co je problém při vícebarevné detekci? •K. Souček Bi9393 Analytická cytometrie •Emission Spectra–Spectral Overlap EmissionSpectra Kompenzace fluorescenčního signálu při vícebarevné detekci n n n n n n nProces při kterém dochází k eliminaci všech fluorescenčních signálů kromě signálu z fluorochromu který má být na příslušném detektoru detekován nNastavení pomocí mixu mikročástic či buněk označených/neoznačených příslušnými fluorochromy. • •K. Souček Bi9393 Analytická cytometrie Co je problém při vícebarevné detekci? •K. Souček Bi9393 Analytická cytometrie Kompenzace fluorescenčního signálu při vícebarevné detekci n n n n n n •K. Souček Bi9393 Analytická cytometrie •“Bright” = good resolution sensitivity •Various fluorochromes-stain index •Choices for 6,- 8,- 10,- and more colors •Fluorochrome selection considerations •FITC Spillover • •650nm •700nm • •PerCP-Cy5.5 •695/40 • • •500nm •600nm •FITC • 530/30 •Wavelength (nm) •550nm • •PE •585/42 •750nm •800nm • FITC Compensation • •650nm •700nm • •PerCP-Cy5.5 •695/40 • • •500nm •600nm •FITC •530/30 •Wavelength (nm) •550nm •PE •585/42 •FITC Compensation • •650nm •700nm • •PerCP-Cy5.5 •695/40 • • •500nm •600nm •FITC •530/30 •Wavelength (nm) •550nm •PE •585/42 Slide 6 FITC adj FITC comp tab with values •FITC Compensation • • 2Fitc comp 1Fitc no comp 3Fitc biexpo •Dot plot showing uncompensated FITC data •Dot plot showing compensated FITC data • •Biexponential dot plot showing compensated FITC data Kompenzace fluorescenčního signálu n n n n n n •K. Souček Bi9393 Analytická cytometrie •FITC positive & negative •PE negative beads • •#2 Kompenzace fluorescenčního signálu n n n n n n • •K. Souček Bi9393 Analytická cytometrie •FITC positive & negative •PE negative beads •NONE! Kompenzace fluorescenčního signálu n Nastavení kompenzací • •parametr - detektor amp. •FL1 - 544 •FL2 - 434 •kompenzace •FL1 - 1.1%FL2 •FL2 - 17.5%FL1 § značené mikročástice – pro běžně konjugované fluorochromy § značené buňky – pro vitální značení •Effects of Changing PMT Values •FITC Voltage Increased by 5 V •FITC Voltage Decreased by 5 V •Correct Compensation •Which marker for compensation? •Small errors in compensation of a dim control (A) can result in large compensation errors with bright reagents (B & C). •Use bright markers to setup proper compensation. •BD Comp Beads •Always positive • •Bright staining • •Save sample (HIV patients) • •Use the same antibody for compensation and the real experiment •BD Comp Beads • •PBMC were stained as shown in a 3-color experiment. Compensation was properly set for all spillovers •Courtesy Mario Roederer •Fluorescence Minus One Tandemové značky http://www.abcam.com/ps/CMS/Images/Tandem-Dyes_image.jpg Tandemové značky - příklad •Tandems are light sensitive •0 hours •2 hours •22.5 hours •PE •(FL2) • •CD8 •CD3 •PE-Cy5 •PE-Cy7 •Time Sample Left in Light Kompenzace - literatura n Mario Roederer - Compensation in Flow Cytometry n Current Protocols in Cytometry (2002) 1.14.1-1.14.20 John Wiley & Sons, Inc. n n M. Loken, D. R. Parks, & L. A. Herzenberg (1977). Two-color immunofluorescence using a fluorescence-activated cell sorter. J. Histochem. Cytochem. 25:899-907. M. Roederer & R. F. Murphy (1986). Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to EGF endocytosis by 3T3 fibroblasts. Cytometry 7:558-565. S. Alberti, D. R. Parks, & L. A. Herzenberg (1987). A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry 8:114-119. C. B. Bagwell & E. G. Adams (1993). Fluorescence spectral overlap compensation for any number of flow cytometry parameters. in: Annals of the New York Academy of Sciences, 677:167-184. •K. Souček Bi9393 Analytická cytometrie Aplikace průtokové cytometrie •ANALÝZA NUKLEOVÝCH KYSELIN •buněčný cyklus a ploidyta •analýza zlomů DNA •inkorporace BrDU •exprese cyklinů •analýza denaturace DNA • •ANALÝZA BUNĚČNÉHO FENOTYPU •imunofenotypizace pomocí CD antigenů •(detekce diferenciačních a nádorových markerů) •detekce cytokinových receptorů • •CYTOGENETIKA •analýza chromozómů • •STUDIUM BUNĚČNÝCH FUNKCÍ •viabilita •stanovení intracelulárního pH •analýza organel a cytoskeletu •stanovení membránového potenciálu •oxidativní vzplanutí •stanovení intracelulárního Ca2+ •stanovení intracelulárních cytokinů •Natural Killer ligace značených buněk •analýza reportérových genů •K. Souček Bi9393 Analytická cytometrie Biologické aplikace průtokové cytometrie nanalýza proliferace nfluorescenční proteiny •K. Souček Bi9393 Analytická cytometrie Buněčný cyklus [USEMAP] d:\powerpoint\figures\chapter18\1801.jpg d:\powerpoint\figures\chapter17\1716.jpg d:\powerpoint\figures\chapter17\1708.jpg http://i.imgur.com/iq9cbSw.jpg Co je důležité při přípravě vzorku a značení… nPostup přípravy vzorku a značení nelze zobecnit – závisí na typu buněk a konkrétní analýze –suspenze jednotlivých buněk –vitální značení –fixace (etanol, formaldehyd) –permeabilizace (detergenty) –difúze –aktivní transport •Analýza buněčného cyklu •jedna z nejstarších aplikací flow cytometrie, stanovení fáze buněčného cyklu podle množství DNA •průtoková cytometrie je vhodná metoda pro rychlou a přesnou determinaci buněčného cyklu •jednoduchým způsobem je DNA obarvena fluorescenční barvou specifickou pro DNA. •Propidium iodide 4′,6-diamidino-2-phenylindole (DAPI) - dramaticky zvyšují fluorescenci po vazbě na DNA. Je nutná permeabilizace cytoplasmatické membrány . •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. - mohou být používány pro značení viabilních buněk. •K. Souček Bi9393 Analytická cytometrie • • • G2 M G0 G1 s •0 • 200 • 400 • 600 • 800 •1000 G0 G1 s G2 M DNA Analysis •DNA content •Count 2N 4N •Normal Cell Cycle •Purdue University Cytometry Laboratories - propidium iodide - DAPI - Hoechst 33342 - 7-AAD Analýza histogramu buněčného cyklu nnepoužívá se běžná analýza pomocí úseček (regionů) v histogramu nje nutné používat speciální software pro modelovaní analýzu distribuce jednotlivých fází Z:\soucek_gr\Data_Soucek_gr\CHKi\Cell cycle arrest\ModFit_CHK1i cell cycle analysis 2017\20170425\rpt_Worklist_A_Tube_001_012_20170425_124644.fcs.bmp •Cell cycle histogram: gating strategy C:\Users\ksoucek\AppData\Local\Temp\Rar$DRa0.904\High.jpg C:\Users\ksoucek\AppData\Local\Temp\Rar$DRa0.877\high2.jpg C:\Users\ksoucek\AppData\Local\Temp\Rar$DRa0.415\medium.jpg C:\Users\ksoucek\AppData\Local\Temp\Rar$DRa0.454\medium2.jpg C:\Users\ksoucek\AppData\Local\Temp\Rar$DRa0.126\Low.jpg C:\Users\ksoucek\AppData\Local\Temp\Rar$DRa0.282\Low2.jpg •Detekce buněk v synchronizovaném buněčném cyklu •File analyzed: SAMPLE2.FCS •Date analyzed: 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 •Aneuploidie je významný diagnostický marker 450px-Native_American_tobacco_flower Analýza ploidity u vyšších rostlin The image “http://florawww.eeb.uconn.edu/images/byspecies/ALSTROEMERIA_CARYOPHYLLACEA01.JPG” cannot be displayed, because it contains errors. • • •http://en.wikipedia.org/wiki/Image:Native_American_tobacco_flower.jpg •Alstroemeria caryophyllacea •Nicotiana tabacum 400px-Corntassel_7095 •http://en.wikipedia.org/wiki/Image:Corntassel_7095.jpg •Zea mays •endosperm 28 dní po opylení •Cell cycle analysis- limitations Z:\soucek_gr\Data_Soucek_gr\CHKi\Cell cycle arrest\ModFit_CHK1i cell cycle analysis 2017\20170425\hst_Worklist_A_Tube_001_033_20170425_144207.fcs.bmp Z:\soucek_gr\Data_Soucek_gr\CHKi\Cell cycle arrest\ModFit_CHK1i cell cycle analysis 2017\20170425\hst_Worklist_A_Tube_001_025_20170425_134529.fcs.bmp Z:\soucek_gr\Data_Soucek_gr\CHKi\Cell cycle arrest\ModFit_CHK1i cell cycle analysis 2017\20170425\hst_Worklist_A_Tube_001_023_20170425_133821.fcs.bmp Z:\soucek_gr\Data_Soucek_gr\CHKi\Cell cycle arrest\ModFit_CHK1i cell cycle analysis 2017\20170426\hst_Worklist_B_Tube_001_021_20170426_131254.fcs.bmp Z:\soucek_gr\Data_Soucek_gr\CHKi\Cell cycle arrest\ModFit_CHK1i cell cycle analysis 2017\20170426\hst_Worklist_B_Tube_001_024_20170426_132616.fcs.bmp Z:\soucek_gr\Data_Soucek_gr\CHKi\Cell cycle arrest\ModFit_CHK1i cell cycle analysis 2017\20170426\hst_Worklist_B_Tube_001_030_20170426_134946.fcs.bmp •Analýza inkorporace BrdU •K. Souček Bi9393 Analytická cytometrie nbromodeoxyuridin se inkorporuje do DNA namísto tymidinu během S-fáze npo fixaci a částečné denaturaci DNA je možné BrdU detekovat pomocí specifické protilátky značené fluorochromem nv posledním kroku můžeme obarvit DNA n •Analýza inkorporace BrdU •K. Souček Bi9393 Analytická cytometrie Click azide/alkyne reaction http://www.invitrogen.com/etc/medialib/en/images/ics_organized/applications/cell_tissue_analysis/da ta_diagram/750_wide.Par.94243.Image.-1.-1.1.gif Invitrogen Aplikace Click-IT (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 detectied 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. Aplikace Click-IT (Invitrogen) analýza syntézy DNA (proliferace) 02ClickiTAnim01 •Tritiated (3H) thymidine •3H-thymidine 02ClickiTAnim01 • Original method for measuring cell proliferation • Radioactive • Not compatible for multiplexed analyses •3H-thymidine 02ClickiTAnim01 BrdU03 •BrdU (5-bromo-2'-deoxyuridine) •BrdU • •Br • •Br • •Br • •Br 02ClickiTAnim01 • •Br • • •Br • •Br • •Br •BrdU 02ClickiTAnim01missing 02ClickiTAnim01 • • •Br • •Br • •Br • •Br •BrdU 02ClickiTAnim01missing • •Br • •Br • •Br • •Br •BrdU 02ClickiTAnim01missing • •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 EduBlack01 02ClickiTAnim01 •EdU (5-ethynyl-2'-deoxyuridine) •Click-iT™ EdU • • • • 02ClickiTAnim01 •Click-iT™ EdU • • • • • FluorBefore01 FluorAfter01 FluorBefore01 FluorAfter01 FluorBefore01 FluorAfter01 FluorBefore01 FluorAfter01 02ClickiTAnim01 •Click-iT™ Edu • • • • FluorAfter01 FluorAfter01 FluorAfter01 FluorAfter01 • Non-radioactive • No DNA denaturation required • Simplified protocol • Small molecule detection • Multiplex compatible, including • Other antibodies • Dyes for cell cycle analysis Analýza DNA a RNA nPyronin Y vs. Hoechst 33342 n- Pyronin interaguje s ds RNA a DNA ale jeho vazba na DNA je inhibována přítomností Hoechst 33342 nAcridine orange n- při interakci s RNA emituje červené světlo a při interakci s DNA zelené Detekce intracelulárních proteinů v kombinaci s detekcí DNA Detekce mitotických buněk nHistone H3 je specificky fosforylován během mitózy (Ser10, Ser28, Thr11) ndvojité značení DNA vs. H3-P identifikuje populaci buněk v M-fázi http://www.cellsignal.com/products/images/3465_ific_jp_090213.jpg http://www.cellsignal.com/common/cellsignaling.gif Flow cytometry most common applications Viability assays (propidium iodid, 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 characterisation of the cells (CSCs markers, differentiation, …) • IMMUNOPHENOTYPING D:\kuloth\2015\May\27-05-2015\NR_aop1\slides_img\nrrheum.2015.71-f1.jpg Principle: cells are stained with monoclonal antibodies conjugated to various fluorescent dyes and analyzed with 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. Rev. 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 Image result for live/dead fixable • 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 alkyne azide triazol • DNA DAMAGE using γH2A.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 double-strand breaks Pros: in theory simple immuno-staining after fix&perm Cons: DSBs can also be intrinsic, occurring in healthy, nontreated cells, DSBs are formed in the course of DNA fragmentation in apoptotic cells Image result for dna damage gH2A.X An external file that holds a picture, illustration, etc. Object name is nihms5372f1.jpg 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 immuno-staining 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 Workflow •Possible issues •Need of optimization •Incompatibility of Fluorochrome with Click-iT reaction •Permeabilization •Over cross-linked •Insufficient/too high concentration •Sufficient permeability •Antibody/ marker selection •Compatibility with other fluorochromes •Sufficient permeability •Antibody specificity Permeabilization Goal: Sufficient for intracellular markers, gentle for surface markers DNA damage – yH2AX Apoptosis - Cleaved Caspase 3 Surface marker – Trop-2 Trop-2 BV421 Trop-2 BV421 Trop-2 BV421 Trop-2 BV421 85,9 % 75,6 % 75,3 % 59,2 % 78,8 % 34,6 % 25,9 % 19,5 % 92,0 % 75,2 % 80, 8 % 83,8% The best solution: 0,25% Triton x-100 DMSO MG-132 MG-132 DMSO DMSO DNA stain •Violet laser DAPI, Hoechst 33342 • FxCycle Violet, … • •Blue laser Vybrant Dyes, PI, … • •Red laser FxCycle Far Red – 7-AAD Broad spectrum of the dyes Problems: High concentration of dye, no wash Spillover & Compensations • Compensation Antibody conjugates: •anti-rat and anti-hamster Igκ/negative control compensation beads (BD Biosciences), •SpheroTM 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. Example of final set-up Parametr Marker Fluorochrome Cell Surface Marker CD44 APC/Cy7 Cell Surface Marker Trop-2 AF488 Viability LIVE/DEAD kit Yellow DNA synthesis Click-iT EdU AF647 Cell Cycle DNA content PO-PRO-1 DNA damage yH2AX PE Apoptosis Cleaved Caspase 3 AF494 Surface Markers DNA synthesis DNA damage Apoptosis Viability Cell Cycle Flow Cytometric Multiparametric Assay was established Examination of small subpopulation (Trop-2+) in response to experimental treatment Detekce počtu buněčného dělení http://www.lifetechnologies.com/content/dam/LifeTech/migration/en/images/ics-organized/applications /cell-tissue-analysis/data-chart/180-wide.par.32782.image.-1.-1.1.gif http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn ology/flow-cytometry/data-images.Par.85351.Image.gif?direct=1 Applied Biosystems The Nobel Prize in Chemistry 2008 n"for the discovery and development of the green fluorescent protein, GFP" Nobel Prize® medal - registered trademark of the Nobel Foundation •http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/ Fluorescenční proteiny nbioluminescence resonance energy transfer (BRET) –Aequorea victoria - medúza žijící ve vodách na pobřeží Severní Ameriky. –je schopna modře světélkovat (bioluminescence). Ca2+ interaguje s fotoproteinem aequorinem. –modré světlo excituje green fluorescent protein. –Renilla reniformis – korál žijící ve vodách na severním pobřeží Floridy. –luminescence vzniká degradací coelenterazinu za katalytického působení luciferázy. –modré světlo excituje 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 Fluorescenční proteiny n Osamu Shimomura –1961 objevil GFP a aequorin n •http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm nScience. 1994 Feb 11;263(5148): nGreen fluorescent protein as a marker for gene expression. nChalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. n nDepartment of Biological Sciences, Columbia University, New York, NY 10027. n n 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. •Fluorescenční proteiny nDouglas Prasher nMartin Chalfie mice_400x300 [USEMAP] Fluorescenční proteiny •http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm in vivo molekulární vizualizace •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 Fluorescenční proteiny nSergey A. Lukyanov –Objevil „GFP-like“ proteiny u nesvětélkujících korálů n Roger Tsien n~ 2002 – mutace FP = barevné spektrum nhttp://www.tsienlab.ucsd.edu/ http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE%20-%20PLATE%20-%20Beach.jpg The image “http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE%20-%20Rendered%20GFP%20-%20640.jpg” cannot be displayed, because it contains errors. Roger Y. Tsien •Detekce buněk v synchronizovaném buněčném cyklu Licensing control by Cdt1 and geminin http://jcs.biologists.org/content/joces/125/10/2436/F9.large.jpg?width=800&height=600&carousel=1 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, inhibitor of DNA replication inhibits Cdt1 •S, G2, M- SCFSkp2 •substrate: DNA replication factor Cdt1 – key licensing factor • •Fucci sensors - 1st generation, coral FP •monomeric Kusabira orange 2 – hCdt1 (30/120) •Monomeric Azami-Green – hGeminin (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) • Image result Fucci http://www.amalgaam.co.jp/products/advanced/img/fucci/E3.jpg http://cfds.brain.riken.jp/Fucci.html CONTROL SCH900776 MU380 …lot of questions, but how to answer them? nHow many times cells divided? nWhat is a length of cell cycle phases? nIs there a difference in time between first and second division? nHow it is all affected by my drugs? SOLUTION (Milan_TrackMate_(Fiji) + D:\Kaja\Desktop\logo_CELLIM_CF_final.jpg Branches (dvisions) analysis 02_02_01_01 Median 14 hours 02_02_01_01 •shRNA for TTL •Ventura, Meissner, Dillon, McManus, Sharp, Van Parijs, Jaenisch and Jacks . •PNAS 2004 “Cre-lox regulated conditional RNA interference from transgene”. •shRNA elements: • •TTL-1 • •tgcatcaaataagcatgagattccaagagatctcatgcttatttgatgcttttttcacgtagtttattcgtactctaaggttctctagagtacgaata aactacgaaaaaagagct • •TTL-2 • •tggcaacgtttggattgcaattccaagagattgcaatccaaacgttgccttttttcaccgttgcaaacctaacgttaaggttctctaacgttaggttt gcaacggaaaaaagagct • • • •Pz-HPV-7 cells - shRNA for TTL •(Lentivirus infection) biarsenical–tetracysteine system nNefluorescenční, membránově permeabilní biarsénová značka vytváří kovalentní fluorescenční komplex s jakýmkoliv intracelulárním proteinem obsahujícím krátký tetracysteinový motiv (CCPGCC) biarsenical–tetracysteine system Shrnutí přednášky nanalýza proliferace nfluorescenční proteiny •Na konci dnešní přednášky byste měli: 1. 1.vědět jakým způsoben je možné analyzovat buněčný cyklus. 2. umět navrhnout další parametr kombinovatelný s DNA analýzou. 3. znát příklady buněčných funkcí které je možné analyzovat na průtokovém cytometru. 4.vědět co jsou to fluorescenční proteiny a jaké jsou výhody jejich využití v buněčné biologii. 5.co je to click-IT. K. Souček Bi9393 Analytická cytometrie