asmV aao* atntrBet,Ig enee-ern lsadaCmi alG idrand Figdor G. Carl Cambi, Alessandra Reinieren-Beeren, Bogaart Inge den Beest*, van ter Geert Martin sampling Baranov*, V. Maksim facilitate cells dendritic of ARTICLE RESEARCH ß 1052 2013 December 9 Accepted 2013; August 21 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This work. { this to equally contributed 6525GA authors 28, *These Grooteplein Geert Netherlands. Sciences, The Life Centre, Nijmegen, Molecular Medical for University Institute Radboud Radboud , only Tumor of Because Department resting 2005). in response al., immune primary et a major induce (Banchereau McGreal can T-cells cells by 1998; dendritic prime the Steinman, cells to antigen dendritic migration and dendritic where the finally nodes, and of of lymph of molecules upregulation maturation co-stimulatory molecules, presentation specific (MHC) in surface of complex . results histocompatibility uptake and or trigger antigen can endocytosis cells of as lectins, receptor-mediated C-type antigen well Recognition of as via as Binding such origin. antigen bacteria, PRRs, viral some or gram-negative pathogen-associated fungal to of bacterial, from recognize of membrane peptides , as outer that such the (PAMPs), Takeda express PRRs cells patterns 2005; Dendritic molecular (PRRs) different al., 2007). al., et receptors et many Kaparakis McGreal 2005; recognition 2002; Akira and Masten, pattern other and so-called and for (Lipscomb presentation. spleen by and the antigen tissue sample in is constantly localized and cells tissues are non-lymphoid dendritic cells of dendritic Immature function main The INTRODUCTION , endocytosis Dendritic Receptor-mediated , presentation, Antigen WORDS: KEY of podosomes the for antigen data sampling these cells. Our antigen localized dendritic T-cells. in role activate to trigger novel protrusive to a can demonstrate presentation locate into and they processing evolve receptors where uptake, the can in structures recognition resistance they protrusive physical Pathogen where human low spots) structures. of of spots podosomes (soft to that substrate network locate show cells we of dendritic Here, and degradation core localized matrix. via actin-rich migration extracellular cell adaptor an facilitate They of adhesive , filaments. consist by mechanosensitive that surrounded podosomes contain called cells acquired an structures and innate play Dendritic between and link antigens immunity. the establishing for in environment role the important sample cells Dendritic ABSTRACT uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,15–04doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. { ait Shite l,21;vndnDise l,2013b). al., et Dries den van 2011; region al., core filaments et actin actin-rich anti-parallel (Schmidt an which radiate of from integrins consist Schachtner by Podosomes 2011; surrounded 2008; Courtneidge, 2013b). al., and al., et Murphy Gimona et 2011; 2004; al., al., et et peripheral Linder Buccione within in migration (reviewed cell tissues facilitate that podosomes of called lymph T-cells. sites draining to the T- specific antigen to present of travel to they to activation where marrow) need nodes for cells bone and dendritic mature tissues), For (i.e. cells, (peripheral systems. origin activity from immune of sampling migrate to adaptive sites need cells and their dendritic immature component innate sampling, crucial the antigen a balances considered are that they T-, naive a ea hr s1t 2mn hra naooi a persist can whereas min, 12 to 1 that as lifetimes al., short with least as dynamic at et very be in in are can Bravo-Cordero invadopodia podosomes involved from First, 2004; differ Although ways. are to two al., considered which 2013a). are et 2009), they al., 2012), (Sabeh Friedl, of invadopodia metastasis and et 2009; well-studied (Wolf al., the Schachtner cells et to Proszynski similar 2011; cancer (Buccione very 2006; are al., al., junction podosomes et the et neuromuscular Helden at van Linder the 2004; such , and al., of cells, cells, et adherent site endothelial of , postsynaptic cells, types , other muscle , many smooth 3D-micropatterned by as also of but 2001) edges 2012). al., the et are Dries Podosomes their den on by (van 2009). substrates demonstrated al., localization as et 2008) al., Dovas selective al., et et 2002; (Zicha (Collin al., cells chemotactic mechanosensitive WASP- et these and 2006). in Jones podosomes al., compromised 1998; form severely et are is not Calle migration do 2001; and podosomes through leukocytes al., cells passage fact, deficient and migrating et migration of (Burns cell In for edge required 2013b). leading is turnover the al., their at located et commonly Schachtner connective 2007; and epithelium facilitates (Matı Protease endothelium, and tissue 2008; through 2011). invasiveness al., migration cell Courtneidge, et promotes cell and Gimona podosomes Murphy extracellular 2006; at 2011; release the al., al., et degrade et Helden locally Linder (van and that (ECM) MT1-MMP) matrix proteases called (also other release MMP14 concentrated several of complex 2002; metalloproteinase points al., the et are nucleating Jones Podosomes of of 2001; actin 2006). al., al., the et the et cores Burns Calle 1999; recruit contain al., actin-rich et can podosomes to (Linder the Arp2/3 that and adhesions, WASP around membrane focal enriched plasma Unlike are the podosomes. and to the connect integrins, and actin , cortical as such proteins Adaptor mauednrtcclsfr atclrcl–arxcontacts cell–matrix particular form cells dendritic Immature oooe r o nyfre ydnrtccls(un tal., et (Burns cells dendritic by formed only not are Podosomes ´ as-Roma ´ ta. 05 amne l,20;Linder, 2007; al., et Carman 2005; al., et n

Journal of Cell Science h ug u n ml netn yetnig‘protrusive (Rescigno extending organs by these of intestine epithelium small the and of lumen through the gut in are dendrites’ antigen cells for lung, al., sampling dendritic of et the capable because (Gawden-Bone be interesting to structures known are well protrusive results MMP14- these These of of 2010). release occurred tips the material the extracellular Both of at pores. uptake filter and the vesicles extend of containing could and filters, lumen pores on the the of cultured into top were on precisely cells formed dendritic podosomes when that demonstrated podosomal allow environment. and extracellular podosomes the might of into but the protrusion morphology 2012), al., dictates al., the only et et affect Dries not also den (Gawden-Bone substrate (van the podosomes filters of of localization polycarbonate geometry the of Thus, 2010). pores the into htpdsmso edii el ol rtuemr hn3 than shown more was protrude it could cells where and of dendritic study of recent depth Murphy podosomes a protrusion that in 2011; limited contradicted is this al., podosomes However, et 2011). (Linder Courtneidge, environment extracellular n a rtuemr hn5 than more protrude can and 2 to 0.4 o or rvee nLne ta. 01 upyand and Murphy smaller 2011; are al., 2 podosomes et to 0.5 Linder Second, only measure in 2011). (reviewed Courtneidge, hours for ARTICLE RESEARCH ,btclswr rw nplcroaefleswt oeszso 0 m h itr eesae ngltncnuae oAeaFur63(itr gra 5 (Filter, Quantif bars: 633 (C) Scale Fluor surface. green). Alexa filter vinculin, to the magenta; conjugated of (actin, location (E) in approximate nm (mean soaked the B 400 were panel indicates of filters from arrowhead sizes pores The red pore filter nm. The the with (means 400 to views. filters cores of podosomes orthogonal actin sizes the the of AB-positive pore gree of alignment of with (AB, positions the percentage filters 488 the the polycarbonate Fluor indicate show on Alexa lines immediately graphs grown to Yellow podosomes Bar were with conjugated cells podosomes. cells antibodies representative chosen but secondary of randomly A, images and confocal indicate paxillin, are arrowheads and Shown Yellow magenta). talin surface. (Phal, vinculin, phalloidin–Alexa-Fluor-564 substrate. against with the stained from directed was resistance antibodies Actin physical primary low with of stained spots to and locate Podosomes 1. Fig. h td yGwe-oee l Gwe-oee l,2010) al., et (Gawden-Bone al. et Gawden-Bone by study The m ,weesivdpdacnrah8 reach can invadopodia whereas m, m ndaee n rtueol about only protrude and diameter in m m rmtecl ufc nothe into surface cell the from m m ndiameter in m 6 ...(,)Shmso edii el ihnnporsv oooe ngas()and (D) glass on podosomes non-protrusive with cells dendritic of Schemes (D,E) s.d.). m m osiue e ehns fhwclsmgtb bet sense to able barriers. be physical might through cells signals how this extracellular and of presentation, mechanism antigen new in a podosomes results where constitutes for Our A structures role antigen. direct protrusive substrate. of a uptake these show the receptor-mediated to within trigger locate can deeper PRRs they from human of the antigen range of help and for wide actin-rich that podosomes sample structures 2010) protrusive to these that into cell al., evolve of demonstrate can et cells we dendritic characterization (Gawden-Bone Here, molecular structures. al. a et performed described first Gawden-Bone structures podosome-like 2013). by protrusive al., be the et might Vallon- to Strisciuglio sampling antigen 2006; related 2013; trans-epithelial al., this al., et that 2012; speculated et Shan al., We et 2013; Chieppa Thornton al., 2012; 2005; et al., et Farache al., Lelouard 2006; et al., et Niess Eberhard 2001; al., et nern n nernascae rtis uha icln talin vinculin, as such proteins, -associated and integrins of van of height 2001; dome-shaped core a as actin al., appear an et glass with on structures (Burns actin-rich Podosomes 1A) 2006). al., (Fig. et podosomes they Helden substrate, of glass a clusters on cultured form substrate are cells the dendritic of human spots When soft in extend Podosomes RESULTS m m. A ua-ooyedrvddnrtcclsclue ngassubstrate glass on cultured cells dendritic Human--derived (A) ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal , 0 m uruddb 5-mwd igof ring wide 250-nm a by surrounded nm, 500 , 5 mdaee and diameter nm 350 6 ...()Sm as Same (B) s.d.). cto of ication tteglass the at n). 1053 y).

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Fig. in to similar magenta) (Phal, phalloidin with visualized was actin and (green) vinculin for immunostained were Cells m. -ie oe Fg A and 2A) (Fig. pores m-sized , 10- m hcns.The thickness. m m .()Qatfcto ftefato fatncrsainn ihtefle oe rmpnl A–C panels from pores filter the with aligning cores actin of fraction the of Quantification (D) m. m A edii el utrdo oyabnt ebaeflesipentdwt Alexa-Fluor-633- with impregnated filters membrane polycarbonate on cultured cells Dendritic (A) oe ntefle wiearw)adtefraino oooe nbt ie ftefle (yellow filter the of sides both on podosomes of formation the and arrows) (white filter the in pores m 00 aendee l,21) hsrslsi rwho a of for growth (i.e. in point 3 some results at above This and pores 2010). structure al., al., podosome-like et protrusive et (Gawden-Bone extracellular forces Labernadie of mechanical degradation 2010; exerting a local by such by and When can substrate matrix and pores). the invasive filter into increasingly become protrude the podosomes (e.g. found, physical is substrate low place of the spots soft in for resistance search cells pores. dendritic the of podosomes of lumen the as such into proteins, protrude adaptor progressively pores, can larger in vinculin, and, core nm actin 400 the from pores, sizes cultured reported pore 3 cells as increasing to for with 2C,E), Thus, filters (Fig. 2010). polycarbonate pores on al., filter et the (Gawden-Bone of migration through previously facilitate cell to the enough whole of large was edges the size the pore at the and distinguished pores be could podosomes individual oe Fg D.O itr ih3- with filters the On between in 2D). present colocalized (Fig. also increasingly exclusively were pores not but were pores the podosomes with sizes, pore larger nw ob seta o hstasnohlu migration transendothelium this for the essential of be well- Secretion are to podosomes 2012). of known turnover Vestweber, and MMP14 2011; blood metalloproteinase immune cross the (Muller, to activate to have order system barriers. in leukocytes endothelium physical blood lymphatic and through other vessel and cell cells the Dendritic of transmigration facilitates oe hnclswr utrdo itr ihlre oeszsof sizes pore larger with the filters of 1- on 2 lumen cultured the were the into cells into when protrude pores protruded to increasingly seem vinculin not However, did and pores m vrl,apcuei mrigweeteiiilnon-protrusive initial the where emerging is picture a Overall, n 3 and m m ,temrhlg ftepdsmsdfes nsmaller in differs; podosomes the of morphology the m, ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal m m. m ndaee Fg BC.O itr ihthese with filters On 2B,C). (Fig. diameter in m m ndaee;Gwe-oee l,2010) al., et Gawden-Bone diameter; in m m n hwn h rtuinof protrusion the showing and m m -ie oe,small pores, m-sized h ih-adpanel right-hand The m no actin of on -ie pores. m-sized ae ythe by cated

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PIP al., of presence et the demonstrating core, Bogaart actin-rich the surrounding den (van PH) 6 .. he needn eet) cl as 5 bars: Scale repeats). independent three s.d., y aeaig.Terdarwed ntegahmr nest ek at peaks intensity mark graph the on arrowheads red The -averaging). ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal b )(i.3) neetnl,porsv podosome-like protrusive Interestingly, 3A). (Fig. 1) , , 09%o oooe) otie igof ring a contained podosomes), of 70–90% 03%o l oooe) u o always not but podosomes), all of 10–30% slr 03,adPIP and 2003), ¨ssler, 2 d ,wihstimulates which ), m P tie ring a stained -PH m. a M 2 b cuuae at accumulates )adITGB1 and 2) ecie nD in described rrowheads so the of ns 1055 w 2 by d 2 -

Journal of Cell Science iia aet hto oooe fclsclue nglass on cultured cells 2013). al., of et Tsujita podosomes 2009; of al., et that (Dovas 3D,E) to (Fig. rate at protrusions similar actin-rich the of a disassembly in resulted with 2004), cells (Peterson al., WASP the et of of inhibitor treatment reversible because and Jones selective 2006), 2001; a al., wiskostatin, al., et et Calle Burns 2002; 1999; al., still al., et structures et protrusive (Linder WASP Podosome-like on 3C). depended (Fig. region this in ARTICLE RESEARCH admycoe ci oe.Terdarwedidctsteapoiaesraeo h itr D aea ,btfrtbln ro asso h spre the show bars Error tubulin. for but 5 C, bars: as Same Scale 1056 arrowhead (D) experiments. yellow filter. independent por The the view. two with of orthogonal least filters surface the at membrane of approximate from and positions the the cells glass indicates indicates multiple on arrowhead line for cultured red yellow data The cells The cores. green). dendritic (AB, actin of 488 chosen (right) intensi Fluor randomly Fluorescence quantification Alexa (B) to and fluorescence. conjugated (left) in antibodies images increase phalloidin–Alexa-Fluor-633 secondary Confocal an with and (C) stained fluorophore A. was FITC in 1 the Actin marked of green). self-quenching lines , of dashed (DQ loss the in gelatin results in degradation collagen Collagen FITC-labeled tubulin. magenta). and double-quenched MMP14 with contain impregnated structures were protrusive Podosome-like 4. Fig. from of extension observed. different pores not the was is filter where cores podosomal glass, the again the on into of tubules protrusion cultured the lumen cells tubulin of the podosomes This into 4D). these extended (Fig. of ring and F-actin the structures penetrated structures, clearly traffic podosome-like microtubules protrusive also of because tip could the vesicles to microtubules Our MMP14-containing via 2010). the that al., to et suggest microtubules (Schoumacher via cells data al., cancer travel of et to invadopodia and (Wiesner of 4C). 2011) tip manner (Fig. al., et -mediated Cornfine 2010) intracellularly a 2010; traffic in al., to microtubules et known on the are (Wiesner from vesicles excluded cores MMP14-containing seemed rather podosome but where actin-rich enriched glass, not on cultured was cells MMP14 of podosomes structures (non-protrusive) podosome-like the that protrusive 1- immunocytochemistry the on by in (formed observed present We was 2010). MMP14 (Gawden- in results al., previous et with collagen Bone agreement in FITC-labeled 4A,B), (Fig. were double-quenched gelatin filters ( the with the when by intensity impregnated fluorescence apparent increased as local MMP14 2011; structures fold) place al., took podosome-like as et ECM protrusive of Linder such degradation 2008; at Indeed, al., 2013b). metalloproteinases, al., et et Gimona of Schachtner 2004; al., release et local (Buccione the on m erdto fEMocr tpdsmsadi dependent is and podosomes at occurs ECM of Degradation .Atnwssandwt hlodnAeaFur56(aet) n h ealpoens M1 a aee yseii oolnlatbde an antibodies monoclonal specific by labeled was MMP14 metalloproteinase the and (magenta), phalloidin–Alexa-Fluor-546 with stained was Actin m. m -ie oe)adti sacerdfeec from difference clear a is this and pores) m-sized , 2- A ofcliae fdnrtcclsclue nfleswt 1- with filters on cultured cells dendritic of images Confocal (A) m napeiu td Gwe-oee l,21) paeof uptake accordance In 2010), microscopy. of electron al., by structures shown et podosome-like was cells protrusive (Gawden-Bone dendritic at study material previous extracellular a sampling antigen in In involved are structures podosome-like Protrusive yorosrain ihmnlyr fteeihla elline cell al., epithelial et the 2006; of Farache monolayers 2012; confirmed al., with as al., and observations 2013) et al., our et et Vallon-Eberhard Strisciuglio by Thornton 2013; al., 2012; 2006; et Shan 2013; al., al., al., et antigen et et Niess Lelouard for 2001; Chieppa sample al., et can 2005; (Rescigno clathrin cells membranes protein epithelial dendritic it across that coat that Given the 5A–C). well-established (Fig. that is structures protrusive found these we to localized observation, this with ieadls yai.W etadesdterl of role the of addressed structures next podosome-like We protrusive cells. these dynamic. dendritic when protru- at protein less more that and endocytosis increasingly and morphology the become conclude their of they sive as pores), we resistance change filter physical Dries composition together, the low den (e.g. van of Taken 2010; substrate spots al., S2C–E; 2013a). encounter et podosomes Fig. Labernadie al., material also (see et (supplementary 2) glass 1, Movies on podosome- cultured protrusive the cells of reduced Moreover, cores were substrate. actin structures glass like the on of podosomes for undulations h periodic 1 than less compared hours to several podosome- exceeded that protrusive lifetimes have because structures invasive, like stable more more become and to they dynamic 2013a). less when proposed al., become et to of Dries seem are den podosomes properties van Indeed, 2013a; core mechanosensitive al., et and actin-rich (Schachtner protrusive podosomes the the to of contribute undulations periodic m. h usrt nlecstelftm fpdsms and podosomes, of lifetime the influences substrate The ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal , -odcmae opdsmsof podosomes to compared 2-fold m oesz that size pore m ypoie of profiles ty (Phal, ie of sizes e indicate s dof ad d

Journal of Cell Science EERHARTICLE RESEARCH lcrnmcocp smlrt adnBn ta. 2010), al., et Gawden-Bone a observed to we (similar where The structures microscopy transmission 5D–F). by protrusive followed (Fig. electron these labeling immunogold at substrates by CD206 confirmed was and CD71 glass DC-SIGN and of non- on PRRs to presence not these podosomes but of structures, protrusive All also podosome-like TFRC) protrusive receptor. We as to endocytic known localized MRC1)]. general (also as CD71 a receptor as known transferrin the the (also for and stained CD206 CLEC7A) as receptor (also known (also DCIR mannose dectin-1 CD209), CLEC4A), as lectin as known C-type known (also [the DC-SIGN PRRs receptors various 1- family monocyte-derived for with cultured immunostaining filters performed We on structures. cells dendritic protrusive located protrusive these uptake we antigen the in possibility, to involved this at PRRs test whether occur determined to first order might In 2 uptake structures. bars: podosome-derived Scale and that experiments. hypothesized independent recognition we two S1E), least antigen at Fig. C Dectin-1, from material DCIR, cells per (supplementary DC-SIGN, multiple for the Caco-2 immunostaining for of with data now Quantification of but (C) A–C, spread surface. as the Same filter (D–F) ortho show approximate B. the bars and the of A Error position indicates in CD71. the described arrowhead and indicates experiments red line the The from yellow cores The cores. actin gray). actin-rich clathrin-positive (Filter, selected gelatin randomly Alexa-Fluor-633-labeled with indicate impregnated arrowheads were filters The green). receptors. (AB, recognition pattern 1- contain with structures filters podosome-like Protrusive 5. Fig. nie Cmie l,20) p2 salgn o DC-SIGN for dots ligand quantum gp120-coated a these is and gp120 2000) al., 2007); et al., (Geijtenbeek prototype et a as (Cambi gp120 antigen envelope HIV-1 the to tethered protrusive to contact the localize structures. that determined in PRRs podosome-like subsequently these membrane We of (i.e. activity 6). sampling cells (Fig. antigen surface) the filter of the membrane to with compared ventral structures protrusive the the at at beads gold of enrichment epromdfntoa paeeprmnso unu dots quantum of experiments uptake functional performed We m oesz B.Atnwslbldwt hlodnAeaFur56(hl aet)adcahi a iulzdb immunostaining by visualized was clathrin and magenta) (Phal, phalloidin–Alexa-Fluor-546 with labeled was Actin (B). size pore m , -od(CSG)to (DC-SIGN) 3-fold m oeszsfr1hu and hour 1 for sizes pore m , -od(CD206) 9-fold ol eihbtdb h APihbtrwsottn(i.8C), protrusions (Fig. wiskostatin podosome-like inhibitor OVA WASP of or the uptake by 2007), inhibited podosomes Moreover, al., be S3A). could et form Fig. (Cambi material not medium (supplementary growth gp120- do up the take but from can cells dots protrude; CHO–DC-SIGN heterogeneously quantum These cells dendritic cannot 8A). of CHO (Fig. instead but when used cells were (2) form DC-SIGN and 400- recombinant podosomes expressing a 8A,B) with (Fig. (where filters 1B) on size Fig. cultured pore were protrusive cells reduced nm dendritic of strongly the presence was when the uptake (1) by because promoted structures, was podosome-like filters the through uptake independent that 7G). and indicating (Fig. 2, endocytosis both 2011) Pitstop via of endocytosis, occurred al., inhibitor 2012) al., et the et by (Dutta Kleist OVA suppressed (von structures be of protrusive could clathrin-dependent uptake actin-rich Uptake the that 7F). at (Fig. occurred showed microscopy through filter imaging the OVA fluorescence measured through of Live-cell by We and 7D–F). filters (Fig. dots 7A–C). gelatin-impregnated (Fig. gp120-quantum both these to of dots permeable uptake gp120-quantum poorly gelatin- cellular only that and or demonstrated not OVA (Burgdorf experiments were CD206 filters Leakage receptor impregnated 2006). mannose performed the al., also for et We ligand well-characterized 2007). the and a to al., (ovalbumin), antigen comparable et OVA is with (Cambi which experiments viruses nm, uptake 40 many about of of size size particle a have motnl,utk fbt p2-unu osadOVA and dots gp120-quantum both of uptake Importantly, AB ofcliae fdnrtcclsclue ngas()adon and (A) glass on cultured cells dendritic of images Confocal (A,B) ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal m m. oa iw Yellow view. gonal etg of centage 1057 D206

Journal of Cell Science ( EERHARTICLE RESEARCH ol ciaeTcls iia ohmndnrtccls these cells, 1058 dendritic human to podosomes Similar via T-cells. antigen determine OVA activate to up took for could (BMDCs) that marrow cells required bone dendritic whether mouse acidification from dendritic vesicular with isolated the experiments cells performed the prevents We proteases. endosomal/ of thereby blocks activation of and Bafilomycin acidification ATPase this the compartments. that on lysosomal demonstrated dependent bafilomycin was intensity. fluorescence with processing increased experiments an Control and of quenching loss in by fluorescent results OVA compartments double-quenched endosomal/lysosomal this in of proteases double-quenched Degradation of 8D,E). fluorescence (Fig. in proteases, OVA increase by the processed from subsequently apparent also as but endocytosed filters, the were through gp120) biotin- without 8A,B). (Fig. (2) dots gp120-quantum (i.e. than and effectively less CD206) dots substantially and quantum DC-SIGN sugar to treated the of with binds inhibition Uptake (which competitive (1) gelatin. by mannan because blocked specific, degrade and be antigen to filters could was uptake able the OVA still and through dots were antigen gp120-quantum conditions, cells of structures control the leakage these because protrusive residual decreased, in was blocked of the uptake completely because antigen not of of was rate uptake disassembly the Although the 3D,E). (Fig. causes which ( indicated as E of Magnifications (F) per beads gold of number the of Quantification (D) marked (B, beads. membrane gold ventral the of structures. Details (B,C) podosome-like 1- protrusive with of filter microscopy gelatin-impregnated electron Transmission 6. Fig. onsfridvda el n h en r hw ( shown are means the and ( cells individual for points P P 5 5 h nie a o nytknu ytednrtccells dendritic the by up taken only not was antigen The .0,totie ardStudent’s paired two-tailed 0.002, .05 w-aldpie Student’s paired two-tailed 0.0005, m oesz n muogl aee o CSG a uln ftecl ssont h pe-ih ftemicrograph). the of upper-right the to shown is cell the of outline (an DC-SIGN for labeled immuno-gold and size pore m i , ii t .()Sm sD u o o D0.Ba est a nrae -oda h rtuin eaiet eta membrane ventral to relative protrusions the at 9-fold increased was density Bead CD206. for now but D, as Same (G) ). ts) cl as 1 bars: Scale -test). t ts) E aea ,btnwwt muogl aeigfrC26 n h uln hw ttebto right. bottom the at shown outline the and CD206, for labeling immuno-gold with now but A, as Same (E) -test). i npnlA n faporsv tutr (C, structure protrusive a of and A) panel in 6 ....Ba est a nrae -oda h rtuin eaiet eta membrane ventral to relative protrusions the at 3-fold increased was density Bead s.e.m.). m m. m ntevnrlmmrn bak n h rtuin rd o 0dfeetcls Data cells. different 10 for (red) protrusions the and (black) membrane ventral the in m h iie muto edii el ecudclueo the on culture could we cells (2) dendritic T-cells, ( of of filters sensitivity amount great limited the al., (1) the et to (Shimonkevitz owing (IL)-2 in However, interleukin results 1983). this of and secretion II increased recognize class MHC an T-cells to bound DO11.10 T-cells epitope) (323–339 DO11.10 S3C). OVA hybridoma Fig. antigen mouse OVA material activate with (supplementary could loaded filters were the that via material BMDCs (supplementary Finally, 2006) S3B). al., receptor et Fig. (the (Burgdorf CD206 podosome-like antigen and OVA protrusive for MMP14 vinculin, form 1- contained with to structures filters able on were structures BMDCs mouse esbeunl rcse ydnrtccls hc might which can cells, antigen dendritic this activation. T-cell by and in result processed that elements eventually conclude subsequently podosomal dendritic We of contain be structures 8F). protrusive (Fig. that the cells at cells occur MHC dendritic can containing human uptake compartments antigen in reached II was the partly in class or that than least cells rather indicated at trans the experiments antigen in of colocalization T-cells the out Nevertheless, to was cis. leaked T-cell presented prolonged antigen antigen be to the that that regurgitated during or possibilities medium step the the incubation from exclude up to taken binding), directly non-specific unable of were (because filters we coated gelatin the from A lcrnmcorpso ua edii elclue na on cultured cell dendritic human a of micrographs Electron (A) , ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal 10 ii rmpnlA elwarwed akpstoso lsesof clusters of positions mark arrowheads Yellow A. panel from ) 3 el)ad()teiaiiyt opeeyws OVA wash completely to inability the (3) and cells) m oeszsadteeprotrusive these and sizes pore m

Journal of Cell Science tutrsi h omto ftasellrprsi the in pores podosome-like transcellular invasive of of 2007). formation role al., et the the (Carman endothelium with in findings These agreement structures change. in morphology podosomes the of are and composition dynamic protein less and and protrusive progressively EERHARTICLE RESEARCH eoeigo h C,adwe ri oe r formed are ( pores if size or large when in and result sufficiently can ECM, This 2013b). the of of al., 2004; of et 2010; Schachtner al., release al., remodeling 2011; et Gawden-Bone et al., 2008; et concentrated al., (Buccione et Linder West 2008; MMP14 degrade the al., et locally as Gimona and through such 2010) metalloproteinases, exert matrix al., podosomes et spots, physical extracellular soft (Labernadie low these den forces of At van spots physical substrate. 2010; to the localize al., cellular in Podosomes et the resistance 2012). Labernadie of al., 2008; geometry et al., and Dries stiffness et the (Collin podosomes sense substrate that of and established into sampling to well antigen evolve increasingly respond is to can It contribute podosomes cells. can dendritic that that demonstrate structures we protrusive study, this In 20 ( with arrowheads treated orange filter with DISCUSSION on marked cells cores dendritic actin for three of uptake the (F OVA-647 side of quantification). of other position for the course the ( 8A,B to Time at (red) Fig. time applied (G) time control (see At was 3. in incubation filter. magenta) Movie fluorescence h on (right, OVA-647 material 1 OVA-647 cultured of supplementary of after and increase solution imaged LifeAct-GFP the filt a and with shows gelatin-coated or green) transfected on inset magenta) (Phal, cells cultured left, phalloidin–Alexa-Fluor-488 cells dendritic (Qdot; dendritic with of gp120 of stained imaging to images were linked sizes Confocal pore cells dots (E) different The quantum experiments. with filter. of filters uptake suspension through antigen (C) A the OVA-647 sizes. or of (B) pore Scheme dots (D) quantum impregnation. of assay gelatin Leakage structures. without (B,C) podosome-like filters. the protrusive through by (OVA-647) OVA–Alexa-Fluor-647 uptake Antigen 7. Fig. 6 ...o he needn eet) cl as 10 bars: Scale repeats). independent three of s.e.m. . 1 m ) oooe become podosomes m), A ceaiso h oto xeiet o asv ekg fqatmdt or dots quantum of leakage passive for experiments control the of Schematics (A) m E,2 (E), m m (F). m 00 n,tee ehp h es ci oe rvn the prevent cores actin only dense but the microtubules. core of perhaps al., the entry there, et reach (Wiesner and, podosomes to MMP14- of 2010) periphery seem where the not contact substrates transiently do glass again vesicles This on 2010). containing podosomes al., et from where cells (Schoumacher differs Cornfine cancer established of 2010; well invadopodia podosome-like al., the is in this et to protrusive (Wiesner similar 2011), tips al., the et protrusive the of to facilitat vesicles ring diffraction- likely and actin conventional Microtubules structures 2013b). by the al., et podosome resolved Dries penetrate in den be (van actin microscopy of this optical super-resolution cannot limited distribution in uneven suggest that stochastic an to cores is seems (STORM) recent there also that group case our although from microscopy data actin, microscopy of consist on of to reconstruction podosomes considered cores (non-protrusive) generally are solid from which differs substrates, of edges glass This the with pore. aligns that the structure ring-shaped a forms actin tlata h aeo rtuiepdsm-iestructures, podosome-like protrusive of base the at least At t 5 ,OA67wsapidt h te ieo h itr The filter. the of side other the to applied was OVA-647 0, ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal m iso bak rPttp2negative 2 Pitstop or (black) 2 Pitstop M eieyo MMP14-containing of delivery e i – iii .Tefl aae sin is dataset full The ). Live-cell ) r ih1- with ers n ihor with and 1059 m the m

Journal of Cell Science EERHARTICLE RESEARCH 1060 1- with filters gelatin-coated structures. on podosome-like cultured DC-SIGN protrusive expressing by stably processing cells and CHO uptake Antigen 8. Fig. Gwe-oee l,21)adteeycnhl h elto cell the the help from (i.e. can tips substrate the thereby within protrusive deeper and for from the antigens 2010) for allow sample from al., PRRs antigen et These (Gawden-Bone of structures. uptake PRRs protrusive when of receptor-mediated these that variety shown to a have protrusive, localize We progressively cells. become dendritic podosomes of sampling 2009). antigen al., et Dovas Matı 2006; 2002; 1998; al., al., al., et et well- et Calle Jones (Zicha 2001; the process al., support this et (Muller, further in Burns function podosomes data of system our role and and immune established blood 2012), for Vestweber, leukocytes of crucial 2011; other membranes is (Wolf and endothelial vessels nucleus cells across the lymph dendritic and of of ECM size Migration the through 2013). by to al., limited substrate cell likely et ( the is size complete in threshold which the a 2010), pores exceed Here, for to barriers. allow need physical can through material migrate degradation extracellular protease-mediated the of and podosomes the by exerted ofcliae ytoo he needn xet ( experts independent three or two by images confocal ( percentage the uptake, n oto el rmteeprmn hw nD(tlatfv el ah.()Utk fOA67(aet)b edii el nafle,immunostain filter, a on (means cells OVA-647 dendritic and by MHC (magenta) for OVA-647 positive of compartments Uptake po of (F) compartments quantifications intracellular each). b indicate show cells arrowheads the graphs Yellow five gray). for bar (Phal, least compartments phalloidin–Alexa-Fluor-546 The OVA-647-positive (at OVA-647. with of stained D th fluorescence was in from OVA-DQ Actin shown (green). apparent the II experiment as of MHC-class the Distribution dequenched, (E) from was OVA-DQ A1. cells gray). bafilomycin control (Phal, with and phalloidin–Alexa-Fluor-564 treated with cells stained to was compared fluorescence Actin filter. the 1- through with magenta) filters (OVA-647, gelatin-coated on 5 cultured with cells treated filter dendritic on cells dendritic for uptake nti td,w eosrt la oeo oooe in podosomes of role clear a demonstrate we study, this In forces mechanical the of effects combined the Eventually, 6 ... fclsta oku p2-o itnlbldqatmdt g10Qo n itnQo,rsetvl)wsdtrie yaayi of analysis by determined was respectively) Biotin-Qdot, and (gp120-Qdot dots quantum biotin-labeled or gp120- up took that cells of s.e.m.) , 3 m m iksai bak rcrirol DS;rd (mean red) (DMSO; only carrier or (black) wiskostatin M )(adnBn tal., et (Gawden-Bone m) ´ as-Roma m . oesz n ihutk fdul-unhdOA(V-Q re)adOVA–Alexa-Fluor-647 and green) (OVA-DQ, OVA double-quenched of uptake with and size pore m needn eet;* repeats; independent 3 ´ ta. 2005; al., et n m mor400-nmporesizeandinthepresenceorabsenceof25 P , .2 ** 0.02; A paeasya ecie nFg o ua edii el and cells dendritic human for 7 Fig. in described as assay Uptake (A) o ugs htteeporsv rneihla edie might dendrites findings transepithelial Our protrusive membrane. these epithelial that or the from suggest of cells bacteria 2001) now of side (M) uptake al., (lumen) the apical microfold facilitates et the this specialized 2012); (Rescigno al., in et cells (Lelouard pores the epithelial across transcellular dendrites 2013). of through al., protrusive et junctions extend Vallon- Strisciuglio can 2006; tight 2013; cells al., al., et dendritic 2012; et Shan al., Here, et 2013; Chieppa Thornton al., 2012; 2005; et al., et Farache al., Lelouard 2006; et al., et Niess Eberhard (Rescigno 2001; epithelium intestine al., small et and gut clear lung, across in antigen 2012), of al., podosomes by et recognition (Sage cells. antigen protrusive of dendritic MHC findings has employ probe our in to to it also analogy presented receptors Interestingly, T-cell peptides T-cells contain for activation. that that structures T-cell reported podosome-like in been presentation, antigen result recently after trafficked and, might and compartments be processed this II only subsequently class cannot be MHC Antigen also to can filter). but polycarbonate up, the taken of side other oyo vdnesget htdnrtcclscnsml for sample can cells dendritic that suggests evidence of body A P , .1.()Sm sA u o o V-4.()Tm oreo OVA-647 of course Time (C) OVA-647. for now but A, as Same (B) 0.01). ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal 6 6 ...o he needn eet) D ofcliae of images Confocal (D) repeats). independent three of s.e.m. ...Saebr:10 bars: Scale s.d.). m m. m gml iiefrMCcasI and II class MHC for sitive 2 1 ann fe of h 1 After mannan. afilomycin-treated increased e dfor ed

Journal of Cell Science aiiaeclua irto hog xrclua arxand matrix become only extracellular can not through podosomes structures migration identified, protrusive cellular is These facilitate spot protrusive. a increasingly such resistance mechanical once low a of and, spots provide for environment to extracellular could cells dendritic thus of and bacteria. capability intra-epithelial the phagocytose podosomes for explanation from mechanistic evolved have ARTICLE RESEARCH eai a aee ihAeaFur63b nuaig2 wv gelatin (w/v) 100 2% with incubating PBS by in skin 633 porcine Fluor from Alexa with labeled was Gelatin filters impregnated Gelatin 20 at 1980) al., et II light class Quaranta MHC anti-clathrin anti-human mouse mouse Q5/13; and (v/v), (v/v) (clone 1:100 1:50 at at Sigma) (C1985, SALK) chain anti- mouse (347510, mouse (v/v), CD71 (v/v), 1:250 at 1:100 human Biosciences) at BD mouse (555953, Systems) CD206 (v/v), R&D anti-human (v/v), 1:200 (MAB1859, 1:200 at dectin-1 at Dendritics) anti-human Bioscience) (DDX0180, BD DCIR anti- anti-human (551186, mouse mouse DC-SIGN) (v/v), 1:500 (or at CD209 at Abcam) at human (ab6161, ts2/16) Connect/MilliPore) anti-tubulin (clone Bio rat ITGB1) (v/v), (MAB3328, 1:100 (or anti-MMP14 anti-CD29 mouse (IM2581, (v/v), mouse Bear-1) 1:200 (v/v), or 1:200 (ITGAM at anti-CD11b mouse MAB3060, Coulter) | (v/v), (349 1:100 anti-paxillin anti- at mouse mouse (v/v), Sigma) (v/v), 1:100 at dilution Sigma) 1:200 (T3287, at talin Sigma) immunofluorescence: (V9131, the anti-vinculin for used mouse were antibodies primary following The reagents and Antibodies (BioWhittaker), ml ultra-glutamine U bovine mM (100 fetal 1 in 10% Bio-one), days antibiotics containing 6 Greiner for Technologies) (FBS, cultured (Life serum were medium obtained) healthy 1640 from our consent isolated RPMI isolated (informed 2002). al., monocytes donors et for blood Vries blood (de peripheral coat buffy from a crucial derived from were cells cells is Dendritic dendritic physical human of body Preparation complex METHODS AND the MATERIALS human in the function. other immune antigen of and understanding of cells for podosome-derived dendritic environment of sample from podosomes how for Understanding monocytes antigen role cells. novel of a dendritic constitutes antigen. uptake for structures samples protrusive cell receptor- localized dendritic a in effectively that This can engaged area podosomes search be the Thereby increase antigen. also of can uptake mediated but membranes endothelial otat-a g (H IgG anti-rat goat S 80Uml U (800 CSF eoe ymtao rcptto ftegltn Hydrophilic gelatin. and 2 the 1, (0.4, of sizes was pore dye precipitation different Unbound 3 with temperature. filters methanol membrane room polycarbonate at by min 30 removed for (Invitrogen) ester eodr nioiswr sd otat-os g (H IgG anti-mouse goat used: were antibodies secondary ojgt 65n)wsfo nirgn(111P.Boi rgp120 ImmunoDiagnostics. Biotin streptavidin from (Q10121MP). purchased dot Invitrogen was quantum at B) from The III used F-actin. was (HIV-1 stain was nm) (655 to (Invitrogen) (v/v) conjugate phalloidin 1:200 Alexa-Fluor- of -633-conjugated (Invitrogen). dilutions dye and 488 -546- Fluor Alexa 488-, with labeled all 11008), 0.25 hni 6 tao tro eprtr.A5- A temperature. room and at 70% ethanol in 96% first in washed then were WA) Kent, Sterlitech, from all PCT3013100; n usqetytaserdit 4wl lt n eti B until PBS in kept and plate coverslips culturing. the 24-well cell from for a detached needed into were transferred filters subsequently the incubation, a and solution. and of gelatin ethanol) min the of with 10 top (sterilized on After positioned sheet was parafilm coverslip a a with of together filter top on placed was PBS m nsmay ehv hw htpdsmscnsml the sample can podosomes that shown have we summary, In n 3m ndaee)(C0110 C1210 PCT2013100, PCT1025100, (PCT0413100, diameter) in mm 13 and m m gml 2 1 mhtrcnB ic) L4(0 ml U (500 IL-4 Gibco), B, amphotericin 2 1 nahmdfe,5 CO 5% humidified, a in ) + )(-10)adga nirbi g (H IgG anti-rabbit goat and (A-11006) L) 2 1 eiiln 100 penicillin, m gml 2 1 lx lo 3 succinimidyl 633 Fluor Alexa m 2 gml cnann atmosphere. -containing m rpe f25 eai in gelatin 2.5% of droplet l m gml 2 1 2 tetmcnand streptomycin 1 h following The . + 2 )(A-11001), L) 1 n GM- and ) + )(A- L) sfrg10qatmdt,btnww lcd50 placed we now but assay same dots, phalloidin– the gp120-quantum with performed for we stained the as antigen test was To OVA–Alexa-Fluor-647 actin microscopy. of confocal and uptake by PFA imaged opposite were 4% Cells The with Alexa-Fluor-488. the filter. fixed incubation, 30-minute were the a After cells on 50 gp120. medium with to cells culture conjugated covered dots-655 was with the were quantum filter filled the contacted filters of rings medium side the plastic (upper) the Then, of gelatin. that top on with such placed impregnated and 1 were with inverted filters that on h sizes 2 pore for pre-cultured were nM) cells 200 dendritic of concentration final a 2007). (to al., biotin et free (Cambi of excess molar 10-fold oe otdwt .%gltn el eedsutdb re ultrasound brief a by disrupted were 1 Cells with gelatin. filters 2.5% on with grown coated were pores cells dendritic derived monocyte Human imaged PIP were cells 60 The in a (Olympus, 7.5. with microscope pH glycerol scanning Japan) laser Tokyo, at confocal (v/v) FV1000 buffer in Olympus an phosphate embedding 68% with sodium to (6-hydroxy-2,5,7,8- and mM prior Trolox 200 PBS (v/v) acid) with 0.01% tetramethylchroman-2-carboxylic washed containing for were medium For phalloidin cells mounting the min. Finally, Alexa-Fluor-546-conjugated 30 Subsequently, min. and antibodies 30 temperature. for Alexa-Fluor-488-labeled with antibodies primary room incubated BSA] and secondary at with PBS (w/v) with overnight washed incubated were 3% CLSM-buffer cells CLSM in were with and in PFA blocked cells 4% glycine diluted and 0.1% in min the with mM 5 min 20 permeabilized immunostaining, 15 for for then with PBS fixed were in [PBS and X-100 Cells h Triton temperature. 4 (v/v) room to 1 at for PBS adhered) at cells filters 1000 on cultured were Cells Immunofluorescence niiino nie paewt 25 with uptake antigen of inhibition filter. the of top on OVA–Alexa-Fluor-647 by avidin coupled with 4 dots-655 at min quantum 30 gp120 nM for biotinylated 20 nM mixing with 40 dots, incubated quantum to was gp120 and of conjugation the fixation For assay uptake immediate gp120–quantum-dot 5 and and OVA with min DMSO) 120 and in above. 60 described 30, stock as 15, immunostaining for mM (40 incubation filters by gelatin-impregnated wiskostatin followed or h, glass 1 on for pre-cultured were cells Dendritic podosomes 4% of dissolution with Wiskostatin-mediated fixed PBS, with for phalloidin–Alexa-Fluor-546. twice with incubated washed co-stained K- and buffer were and 7.4, PFA filters mM with pH the 20 DTT, Finally, once mM K-gluconate, 2011). 1 0.5 washed with and mM min s 120 ATP-Mg then 30 1 mM with Hepes-KOH, were 2 pulse s acetate, mM Filters 0.1 20 power, (10% given. containing pulses placed three were was and Sonifier) container filter interval) Digital glass the (Branson large above sonicator a cm the of 3 of bottom tip the PBS The some on with PBS. placed with twice with washed and 2011) were temperature al., cells room et with at filters Bogaart the den brief, In (van modifications. previously described as pulse olgnwsvsaie fe iainadsann o -ci with F-actin Degraded for PBS. staining in and gelatin fixation 1% after in phalloidin–Alexa-Fluor-633. visualized fluorescein) was with collagen conjugated skin ihsalrpr ie f40n,()uigCOclsta stably that cells and CHO cells, using dendritic of filters (4) instead using nm, 2007) (3) 400 al., et gp120, of (Cambi of sizes DC-SIGN instead expressed pore biotin smaller to with conjugated dots quantum C erdto.Hmnimtr edii el eegonfr1h 1 for 2.5 grown with were prepared cells filters dendritic on immature Human degradation. 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T-cell line of hybridoma readout T-cell a the as production, IL-2 measuring assay For activation DO11.10 and ELISA Subsequently, time. without culturing and Caco-2) inserts (for the EA.hy926) of days (for days side 20 10 outer after and formed the were acids, on monolayers amino Confluent non-essential FBS gelatin. 1% 20% glucose, Caco-2, outer and mM line the 25 antibiotics cell with on epithelial DMEM FBS the in For 10% cultured inserts. and the antibiotics of side ultra-glutamine, mM 1 glucose, aceeu .adSena,R M. R. Steinman, and J. Banchereau, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.141226/-/DC1 online available material Supplementary material Supplementary immediate for PMC in Deposited NWO. from Framework award release. Spinoza Seventh a Union’s the of European from and the Grant Programme Advanced under for an Council of Organization Program Research recipient Netherlands Science European the the Frontier is C.G.F. from Human (NWO). grant the Research Meervoud of Scientific a grant of a and of recipient Organization the number agreement is under [grant A.C. Council Programme Research 336479]. Framework European Centre the Seventh Medical from Union’s University Grant European Starting Radboud the a the of recipient from the fellowship is and a by funded is G.v.d.B. manuscript. Funding the on commented and results A.C. G.v.d.B., the M.t.B., wrote M.V.B., discussed microscopy. and C.G.F. electron and experiments the in the assisted performed I.R.B. and paper. the designed G.v.d.B. and M.t.B. M.V.B., contributions Author interests. competing no declare authors The interests Competing Medical University Radboud Mietske (all and microscopy Netherlands). cells electron The Caco-2 the Centre, for with Fransen help Jack for comments, Wijers for Friedl Peter thank standard We using Biosciences) Acknowledgements BD 554426, and procedures. (554424 ELISA anti-IL- antibodies HRP-conjugated production and IL-2 capture to the 2 monoclonal days, adhered in using 6 BMDCs by was for the measured culturing T-cells of was After fraction to cells). a BMDC (1000–10,000 only filter of that the ratio given the 1:20–1:200 of but order used, were cells DO11.10 CSG n D0 epesdb edii cells). for dendritic immunostained by and (expressed fixed were CD206 samples and the DC-SIGN Finally, h. 1 for incubated ucoe . rh .D n cie,M A. M. McNiven, and D. J. J. Condeelis, Orth, and R., L. Buccione, Hodgson, J., J. Bravo-Cordero, Fa and C. Brakebusch, he ie ihPS n O11 el nRM eeaddt the to added were RPMI in overnight. cells incubated DO11.10 and washed and filters were filters PBS, washed The h. with 1 times for above three described as performed was pores) oooe,ivdpdaadcrua oslruffles. dorsal 647-657. circular and invadopodia podosomes, metastasis. during migration and invasion cell affair. love immunity. m ora fCl cec 21)17 0216 doi:10.1242/jcs.141226 1052–1064 127, (2014) Science Cell of Journal gml 2 Nature 1 MOJ. EMBO .Aia tde eeapoe yteAia Ethics Animal the by approved were studies Animal ). 2 1 V-4 nRM a de oteotrcabrand chamber outer the to added was RPMI in OVA-647 392 slr R. ¨ssler, 22 245-252. , 2324-2333. , 20) h nernatncneto,a eternal an connection, integrin-actin The (2003). m gml 19) edii el n h oto of control the and cells Dendritic (1998). 2 1 paeasyo itr (1 filters on assay uptake ) , ur pn elBiol. Cell Opin. Curr. 0,0 MC n 1 and BMDCs 200,000 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 20) otadmouth: and Foot (2004). , 00clswere cells 5000 21) Directed (2012). , 24 000to 20,000 277-283. , 6 10 m 2 b m 5 6 1 - ,

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