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1 Cunnington, AJ; de Souz, JB; Wlther, M; Riley, EM (2012) Mlri impirs resistne to Slmonell through heme- nd heme oxygensedependent dysfuntionl grnuloyte moiliztion. Nture mediine, 18 (1). pp ISSN DOI: Downloded from: DOI: /nm.2601 Usge Guidelines Plese refer to usge guidelines t or lterntively ontt reserhonline@lshtm..uk. Aville under liense:
2 Mlri impirs resistne to Slmonell through hemend heme oxygense-dependent dysfuntionl grnuloyte moiliztion. A. J. Cunnington, J.B. de Souz, R-M. Wlther, E. M. Riley Supplementry Figure 1 Supplementry Figure 1. Plsm hptogloin nd hemopexin levels re not depleted y Py17XNL or phenylhydrzine hemolysis. (,) Plsm hptogloin () nd hemopexin () were mesured y ELISA during Py17XNL infetion nd 18 h fter phenylhydrzine (PHZ) dministrtion. Dt re representtive of 2 independent experiments (men ± s.d. of 3 4 mie) per ondition. Signifine ws determined y one-wy ANOVA with post-ho omprison with ontrol ondition using Dunnett s multiple omprison test. **P <0.01, ***P <0.001.
3 Supplementry Figure 2 Control Py17XNL CD11 Gr-1 F4/80 CD68 Ly6C 7/4 Ly6G CD115 GFP CD11 Gr-1 Ly6G F4/80 Supplementry Figure 2. Definition of myeloid ell popultions y flow ytometry. () Myeloid ell popultions in lood were defined y differentil expression of CD11 nd Gr-1 in oth uninfeted nd Py17XNL-infeted mie (14 d post infetion), nd their identity onfirmed y expression of other surfe mrkers (histogrms). Regions,, (nd filled, solid line nd dshed line histogrms) orrespond to grnuloyte, inflmmtory monoyte nd resident monoyte popultions respetively. () Representtive flow ytometri nlysis of lood 18 h fter infetion with GFP-expressing S. typhimurium on dy 15 of Py17XNL infetion. Grnuloytes were identified y Gr-1 Hi expression (left hnd pnel) nd Ly6G expression (right hnd pnel).() Spleni monoyte nd mrophge popultions were defined y flow ytometry s F4/80 Lo CD11 Hi nd F4/80 Hi CD11 Lo respetively (left hnd pnel) nd the proportion of ll GFP + positive ells in eh of these omprtments ws quntified in Py17XNL-infeted, PHZ- nd hemin-treted mie t the humne endpoint (right hnd pnel). GFP + ells re shown 72 h fter infetion of PBS-treted ontrol mie, ut due to very low infetion levels in PBS-treted mie 18 h fter infetion, resulting in no GFP + ells eing deteted in most mie, this ondition is not shown. Dt re representtive of 2 independent experiments with 3 5 mie per ondition. Signifine ws determined y one-wy ANOVA with post-ho omprison with ontrol ondition (PBS) using Dunnett s multiple omprison test.
4 Supplementry Figure 3. d Supplementry Figure 3. Phgoytosis of S. typhimurium y monoytes nd neutrophils is not impired y Py17XNL, phenylhydrzine or hemin nd S. typhimurium killing is not impired y phenylhydrzine or hemin. (,) Intrellulr teri were quntified y onfol mirosopy following inution of CD11 + ells (isolted from lood of Py17XNL-infeted (dy 14) nd uninfeted ontrol mie) with S. typhimurium for 45 min in gentmiin protetion ssy. () Representtive orthogonl reonstrution from 17 stked imges t 0.5 µm depth intervls, showing CD11 + ell (from Py17XNL infeted mouse, rrow) ontining GFP + S. typhimurium. Blue nuler stining, DAPI; Green, GFP; sle r 10 µm. () Summry dt for nlysis of onfol imges. Dt re representtive of 2 independent experiments (men ± sd s.d. of 3 mie) per ondition. Phgoytosis (,d) nd killing (d) ofs. typhimurium y CD11 + ells isolted from whole lood of ontrol mie or test mie 24 h fter PHZ or hemin tretment, nd inuted with S. typhimurium in vitro in gentmiin protetion ssy. Phgoytosis ws ssessed y the perentge of grnuloytes nd monoytes whih were GFP + y flow ytometry () nd y ulture of ell lystes olleted fter 45 min inution with teri (d). Bteril killing ws ssessed y ulture of ell lystes olleted fter 2 h inution with teri (d). Dt re representtive of 2 independent experiments (men ± s.d. of 3 6 mie) per ondition. Signifine ws determined y two-tiled Student s t-test () or one-wy ANOVA with post-ho omprison with ontrol ondition using Dunnett s multiple omprison test (,d).
5 Supplementry Figure 4 Gr-1 Lo Gr-1 Int Gr-1 Hi FS Gr-1 Rhodmine Supplementry Figure 4. Oxidtive urst tivity is predominntly property of Gr-1 Hi one mrrow grnuloytes. Left hnd pnel: Representtive flow ytometri nlysis of ontrol one mrrow ells showing gting of Gr-1 Lo, Gr-1 Int (Gr-1 intermedite) nd Gr-1 Hi ells. Right hnd pnels: rhodmine fluoresene intensity of unstimulted (shded) nd PMA-stimulted ells (unshded, lk line). Results re representtive of 6 experiments eh with 3 5 mie.
6 Supplementry Figure 5 Supplementry Figure 5. Frequeny nd phenotype of one mrrow nd peripherl lood grnuloytes. Flow ytometri nlysis of lood (upper pnel) nd one mrrow (lower pnel) grnuloytes ssessed y expression of Gr-1 nd divided s low, intermedite nd high expression (Gr-1 Lo, Gr-1 Int, Gr-1 Hi respetively), 22 h fter mie were treted with PBS, PHZ or hemin tretments, or t different time points during Py17XNL infetion, or with dditionl S. typhimurium infetion 16 h (PHZ nd hemin), 18 h (Py17XNL nd PBS) or 72 h (PBS) efore hrvest. Grnuloyte frequenies re shown s perentge of ll leukoytes in lood, nd of ll one mrrow ells, respetively. Dt re representtive of t lest 3 independent experiments (men ± 95% onfidene intervl of 3 5 mie) per ondition nd time point.
7 Supplementry Figure 6 d Grnuloytes Inflmmtory Monoytes Resident Monoytes Non-myeloid Cells Control Py17XNL PHZ Hemin Perentge of mximum HO-1 Supplementry Figure 6. Hemolysis nd hemin use systemi nd ell-type speifi indution of HO-1. Heme oxygense-1 indution ssessed y Hmox1 mrna expression in liver (), HO io-tivity in liver () nd HO-1 protein onentrtion in plsm (). () Hmox1 expression in liver ws determined y rt-pcr expressed s fold hnge reltive to the ontrol gene Gpdh, reltive to the differene etween Hmox1 nd Gpdh expression in ontrol liver. Dt re representtive of 2 independent experiments (men ± s.d. of 4 5 mie per time point). Similr results were otined using Tp s the ontrol gene. () HO enzyme tivity in liver homogentes from mie infeted with Py17XNL or 18 h fter PHZ tretment, ws determined y onversion of hemin to iliruin, stndrdized for protein ontent nd expressed reltive to ontrols t eh time point. Dt re representtive of 2 independent experiments (men ± sd s.d. of 3 11 mie per ondition nd time point). () Plsm HO-1 protein onentrtion ws determined y ELISA. Dt re representtive of 2 independent experiments (men ± s.d. of 4 mie per ondition or time point). (d) HO-1 expression in myeloid nd non-myeloid ells in lood ws determined y flow ytometry, using the ell popultion definitions shown in Supplementry Fig 2. HO-1 expression ws quntified (lower pnels) y the rtio of nti-ho-1 fluoresene (unfilled histogrms) reltive to ontrol ntiody (filled histogrms) (upper pnels) nd normlized to the verge vlue for ontrol nimls in eh experiment. Dt re representtive of t lest 2 independent experiments with 3 9 mie per ondition. Signifine ws determined y one-wy ANOVA with post-ho omprison with ontrol ondition using Dunnett s multiple omprison test. * P <0.05, **P <0.01, ***P <0.001.
8 Supplementry Figure 7 F4/80 + F4/80 CD11 Supplementry Figure 7. Frequeny of F4/80 + ells in one mrrow. Flow ytometri nlysis of the proportion of one mrrow ells stining positively for the monoyte/ mrophge mrker F4/80, during Py17XNL infetion or 22 h fter PBS, PHZ nd hemin tretment. Dt re representtive of 1 or 2 independent experiments (men ± s.d. of 3 11 mie) per ondition nd time point. Signifine ws determined y one-wy ANOVA with post-ho omprison with ontrol ondition using Dunnett s multiple omprison test. * P <0.05, **P <0.01.
9 Supplementry methods Regents. All regents were purhsed from Sigm unless speified otherwise. Hemin (ferriprotoporphyrin IX hloride), tin protoporphyrin IX (SnPP), nd olt (III) protoporphyrin IX hloride (CoPP) were otined from Frontier Sientifi, were proteted from light nd prepred y dissolving in 0.2 M NOH, diluted to the desired onentrtion in PBS nd uffered refully to ph 7.5 with HCl. Hemin solutions were susequently filtered through 0.2 µm rodis syringe filter unit (Pll Corportion) nd the onentrtion of the filtered solution determined using Quntihrom Heme ssy y( (BioAssy Systems) ording to the mnufturer s instrutions. SnPP nd CoPP solutions were not filtered ut were prepred using sterile regents nd tehnique. Aliquots of hemin (3.26 mg ml -1 ), SnPP (3 g ml -1 ) nd CoPP (1mg ml -1 ) were stored t 80 ⁰C until use. Phenylhydrzine hydrohloride 25 mg ml -1 solution ws freshly prepred immeditely prior to injetion y dissolving in PBS, uffering to ph 7.4 with NOH, nd filtering through 0.2 µm syringe unit. Slmonell enteri serovr Typhimurium (S. typhimurium) onstitutively expressing GFP ws gift from Prof. Dvid Holden (Imperil College, London, UK). Bteri were grown to lte log phse in stti ulture in Luri-Bertni (LB) roth with 50 µg ml -1 reniillin. Bteri were frozen in liquots in 10% Glyerol nd stored t 80 ⁰C until required for use. The onentrtion of stok teri ws determined y dilution ultures on LB Agr, nd reonfirmed for eh liquot t the time of use. Prior to inoultion, the Slmonell stok ws defrosted, wshed twie in PBS nd diluted to the desired onentrtion in sterile PBS. For in vitro experiments Slmonell were opsonized in 20% norml mouse serum t 37⁰C for 30 min prior to inoultion. For quntittion of teril lods from orgns of Slmonell infeted mie, ell suspensions were prepred y disruption of tissue with syringe plunger, pssge through 70 µm nylon ell striner (BD), nd resuspension s 10% solution y weight in sterile PBS. 10 fold-dilutions of orgn homogentes nd whole lood were mde in 1% Triton X-100, plted onto LB gr nd inuted for 18 h efore ounting the numer of olony forming units (CFUs). Animls. Animl experimenttion onformed with UK Home Offie Regultions nd ws pproved y Institutionl ethil review. Femle, 6 10 week old C57BL/6 mie were otined from Hrln nd Chrles River, UK nd mintined under rrier onditions. Frozen stoks of lood-stge Plsmodium yoelii 17X Non-Lethl (Py17XNL) were inoulted in pssge mie. Blood ws olleted fter 5-7 dys nd experimentl nimls were infeted y intrperitonel (i.p.) injetion of 10 5 prsitised red lood ells (prbcs). Prsitemi ws determined y exmintion of Giems-stined thin lood smers. Erythroyte ount ws determined using Z2 Coulter prtile ounter (Bekmn Coulter). Prsitemi, erythroyte ount nd, where relevnt, ody weight were monitored t lest lest twie week. To indue ute hemolysis, mie were treted with phenylhydrzine (125 µg g -1 ody weight) y suutneous (s..) injetion. Hemin ws dministered y i.p. injetion (50 µmol kg -1 ody weight per dose) in two doses 12 h prt. Slmonell infetions were initited y i.p. inoultion of 10 5 CFU of S. typhimurium in 200 µl PBS, on dy 15 of Py17XNL infetion or 6 h fter PHZ or first dose of hemin tretment. To inhiit heme oxygense tivity, SnPP (40 µmol kg -1 dose -1 ) ws dministered y ip i.p. injetion 48, 24 nd 8 h efore S. typhimurium infetion of Py17XNL infeted mie, 2 h efore PHZ tretment, or 8 h efore S. typhimurium lone. The HO-1 induer CoPP (10mg kg -1 ) ws dministered i.p. 6 h prior to S. typhimurium infetion. Control nimls reeived injetions of equivlent volumes of PBS. After infetion with S. typhimurium, mie were monitored 6 hourly until displying signs of illness (linil stge 2, Supplementry Methods Tle 1) nd then every 1-2 h until they rehed linil stge 4, t whih point they were euthnized. Sine progression from stge 4 to deth is extremely rpid in Slmonell nd rodent mlri infetions, nd using deth s n experimentl endpoint ws onsidered unethil, the humne endpoint - time of rehing stge 4 - ws used for survivl nlysis. In eh experiment group of PBS-treted Slmonell-infeted mie ws srified t the sme time s the tretment groups to llow omprison of teril lods. In ll experiments with groups of Slmonell-infeted mie, nimls were killed y injetion of pentoritol. In ll other experiments mie were killed with CO 2 inhltion. Immeditely fter deth, under septi tehnique, lood ws olleted y rdi punture into heprinised syringes nd tissues were removed into ie old RPMI nd stored on ie, proteted from light, until proessing. Tle 1. Clinil sle used to determine disese severity in mie: 1. no signs 2. ruffled fur nd/or norml posture nd/or minor weight loss (<15%) 3. lethrgy nd/or moderte weight loss ( 20%) 4. redued response to stimultion nd/or txi nd/or respirtory distress/hyperventiltion 5. prostrtion nd/or prlysis nd/or onvulsions nd/or severe weight loss (>25%) 6. Deth The humne endpoint ws defined s mie rehing stge 4
10 Flow ytometry. Antiodies used re shown in Supplementry Methods Tle 2. For ll experiments exept oxidtive urst ssys, ells were inuted for 5 min with red lood ell lysing uffer (Sigm), wshed, nd resuspended in FACS uffer prior to stining. Cells from S. typhimurium infeted mie were fixed in 4% formldehyde prior to surfe stining. Cells were inuted with oktils of ntiodies for 30 min t room temperture in the drk nd wshed twie efore nlysis. Intrellulr stining for HO-1 ws sed on the method of Ewing et l 56. Briefly, ells were fixed in 2% formldehyde for 10 min t 37⁰C, entrifuged t 500 g for 5 min t 4⁰C, resuspended in ie-old 90% methnol nd inuted on ie for n dditionl 30 min. After wshing with FACS uffer, ells were resuspended in FACS uffer ontining 1% norml mouse serum (Southern Bioteh) nd Mouse F Blok (BD Biosienes) or PE onjugted ntiody ginst CD16/32 nd inuted for 5 or 30 minutes t room temperture respetively. Cells were then entrifuged t 1000g for 2 minutes nd resuspended in FACS uffer ontining rit polylonl ntiody ginst HO-1 or n equivlent onentrtion of norml rit polylonl ntiody s ontrol for 30 min t 4⁰C. Cells were wshed twie in FACS uffer efore resuspension with FITC onjugted seondry ntiody nd oktil of surfe mrker ntiodies nd inution t 4⁰C for 30 min, or room temperture for 90 min when the ntiody ginst CD34 ws used, followed y 2 finl wshes. A BD FACSCliur flow ytometer ws used to quire ll smples exept those for nlysis of one mrrow progenitor popultions (whih were quired using BD LSR-II) nd dt were nlysed using FlowJo version 7.6 (Tree Str, In.). Mirosopy. Chmer slides were proteted from light nd ir-dried for 2 h efore nuler stining nd mounting with DAPI dissolved in onfol mtrix (Miro-Teh-L). Slides were exmined using Zeiss xiovert onfol mirosope with Pln-Apohromt 63x oil immersion lens nd Zeiss LSM510 nlysis softwre. For quntittive ssessment of phgoytosis, ells with overlpping GFP + S. typhimurium were ssessed further y 0.5 µm intervl Z-stk imging to determine whether teri were intrellulr or dherent to the ell surfe. For light mirosopy, thin lood films were fixed with methnol nd ir dried efore stining with My-Grünwld Giems stin ording to the mnufturer s instrutions. Imges were quired using Zeiss Axiopln2 mirosope with CP Apohromt 100x oil immersion lens, nd imges were otined with Retig 2000R mer (QImging) nd nlysed using Voloity softwre (PerkinElmer). Slmonell phgoytosis nd killing ssys. Ex-vivo phgoytosis nd killing of S. typhimurium y lood grnuloytes nd monoytes were ssessed in gentmiin protetion ssy nd quntified y flow ytometry, onfol mirosopy nd teril ulture. Following red lood ell lysis, CD11 + ells were isolted from murine whole lood using nti- CD11 mgneti eds (Miltenyi), ording to the mnufturers instrutions. After wshing twie in DMEM (Gio), ells were resuspended t 5.6x10 5 ml -1 in DMEM without ntiiotis nd seeded t 1x10 5 per well in flt ottomed 96-well pltes or t 2x10 5 per well in 8-well hmer slides (Ltek). Pltes nd slides were inuted t 37 ⁰C nd 5% CO 2 for 20 min prior to ddition of S. typhimurium t multipliity of infetion (MOI) of 10 S. typhimurium: 1 CD11 + ell. To ontrol for utofluoresene nd inding of opsonised S. typhimurium to the ell surfe without phgoytosis, wells ontining uninfeted ells (negtive ontrol), or wells in whih oth ells nd teri were fixed with 2% formldehyde (fixed ontrols), were inuted in prllel. Pltes were inuted for 15 min efore ddition of gentmiin to finl onentrtion of 100 µg ml -1 to kill remining extrellulr teri (ie. those whih hd not een phgoytosed) nd inution for nother 30 min. To ssess teril phgoytosis, fter 30 min ells were wshed twie with wrm sterile PBS nd either hrvested for flow ytometry, fixed in situ for onfol mirosopy, or lysed for teril ulture. Alterntively, to ssess teril killing, ells were wshed twie with wrm medium, then reinuted for 2 h in medium ontining 10 µg ml -1 gentmiin to prevent extrellulr growth of S. typhimurium. To ssess phgoytosis y flow ytometry, ells were gently srped from wells (on ie) nd resuspended in PBS with 2% formldehyde efore stining with APC-onjugted ntiody ginst Gr-1 nd PE-Cy7-onjugted ntiody ginst CD11. Phgoytosis ws quntified s the proportion of GFP + ells fter sutrtion of the proportion of GFP + ells in the fixed ontrol smples. To ssess phgoytosis nd teril survivl y ulture, ells were wshed twie in wrm sterile PBS to remove gentmiin, then lysed with 1% Triton X-100 nd 10-fold dilutions were plted onto LB gr, inuted for 18 h t 37⁰C nd olonies ounted. To ssess phgoytosis y onfol mirosopy, ells were fixed with 2% formldehyde for 15 min t room temperture, then wshed twie in PBS ontining 5% fetl lf serum, nd llowed to ir dry efore stining nd mounting.
11 Oxidtive urst nd degrnultion ssy. Neutrophil oxidtive urst ws ssessed using modifition of the flow ytometri ssy desried y Rihrdson et l µL liquots of fresh whole lood, or one mrrow suspension (ells from 1 femur resuspended in 500 μl RPMI), were mixed with either 50 µl of PMA solution (stimulted smples, 25 µm for whole lood, 2.5 µm for one mrrow) or sterile PBS (unstimulted) nd inuted for 15 minutes in 37⁰C wter th. Next 25 µl of stining oktil ontining dihydrorhodmine 123 nd fluorohrome- onjugted ntiodies to ell surfe mrkers in PBS ws dded nd ells reinuted for 5 min t 37⁰C. 2 ml of red lood ell lysis uffer ws dded to eh tue nd inuted in the drk for 15 min t room temperture then entrifuged t 1000 g for 1 min. Cells were wshed gin with PBS, nd resuspended in PBS with 1% prformldehyde. The mgnitude of the oxidtive urst ws ssessed y rhodmine medin fluoresene intensity (MFI) mesured in the FL-1 hnnel, nlysed on the sme dy. Degrnultion ws mesured y the perentge inrese in the MFI of surfe CD11 expression in stimulted versus unstimulted smples. In eh experiment t lest three ontrol smples (from ge-mthed, helthy, uninfeted mie) were ssyed in prllel with experimentl smples. For longitudinl ssessment of the oxidtive urst nd degrnultion t different time points in the sme experiment, rhodmine fluoresene vlues were normlized to the verge vlue for the ontrol smples t eh time point. Mesurement of heme nd hemogloin. Stndrd hemogloin solution ws prepred y 0.2 µm filtrtion of lysed red lood ell superntnt. The onentrtions of stndrd solutions of hemin nd hemogloin were determined using Quntihrom Heme nd Hemogloin ssy kits (BioAssy Systems) in ordne with the mnufturer s instrutions. Protein ound plsm heme ws quntified y the spetrophotometri method of Shinowr nd Wters 57 using NnoDrop ND1000 spetrophotometer (Nnodrop Tehnologies). Briefly heprinised whole lood ws entrifuged t 500g for 5 minutes, then plsm ws removed nd sujeted to entrifugtion t 15000g for further 7 minutes to pellet ny remining red lood ells. Two µl of the remining plsm ws used to determine sorne t 562, 578, 598, 615 nd 675nm wvelengths. Using the sorption vlues t 562, 578 nd 598nm the onentrtion of plsm hemogloin ws determined y the method of Khn et l. 58. The effet of plsm hemogloin on the differene in sorption nm ws determined y preprtion of stndrd urve of hemogloin in plsm, nd the sorption differene nm ws orreted for the effet of plsm hemogloin. The orreted sorption t nm ws used to determine plsm heme from stndrd urve for hemin in plsm. Hmox1 expression, HO tivity nd HO-1 protein ssys. Hmox1 mrna expression in liver ws determined from fresh snips of liver whih were snp frozen in liquid nitrogen nd stored t 80⁰C until proessing. RNA ws extrted (RNAesy, Qigen) nd DNAse1- treted prior to DNA synthesis. DNA ws quntified using pre-vlidted inventoried Tqmn gene expression ssys for Hmox1 (Mm _m1), Gpdh (Mm _g1) nd Tp (Mm _m1), nd n ABI Prism 7000 sequene detetion system (Applied Biosystems). DNA expression for eh smple ws stndrdized using the housekeeping genes Gpdh nd Tp nd expressed s reltive fold hnge ompred to helthy ontrol smples. HO tivity ws mesured in whole liver homogentes fter RBC lysis, using the method of Foresti et l. 59. To llow omprison etween experiments, HO tivity ws normlized to the verge vlue of t lest three ontrol smples in eh experiment. Plsm HO-1 ws mesured y ELISA using HO-1 Immunoset (Assy Designs) performed in ordne with the mnufturer s instrutions. Sttistil nlysis. Sttistil nlysis ws performed using Grph Pd Prism 5 softwre. All sttistil nlyses were pre-plnned nd used n lph vlue of Survivl nlysis ws performed using the Log Rnk Mntel Cox test. Continuous dt whih were pproximtely normlly distriuted were nlysed using two-sided unpired or pired student s t-test for pirwise omprisons, or one-wy ANOVA with post-ho testing using Dunnett s multiple omprison test for omprison with the ontrol group, Tukey s multiple omprison test for omprison etween multiple groups, nd Bonferroni s multiple omprison test for omprison etween two or more seleted pirs. All dt relting to teril lods were log 10 -trnsformed prior to nlysis. Comprison of proportions etween groups ws performed using Fisher s ext test.
12 Supplementry Methods Tle 2: ntiodies used for flow ytometry Antigen (lone) Mnufturer Fluorohrome Gr 1 (RB6 8C5) ebiosiene FITC, PE, PE Cy7, APC, efluor 450 CD11 (M1/70) ebiosiene PE, PE Cy7 Ly6G (1A8) Miltenyi Biote APC F4/80 (BM8) ebiosiene FITC, APC CD68 (FA 11) ADSerote AlexFluor 647 Ly6C (Hk1.4) Am FITC Biolegend APC Ly6B.2 (7/4) ADSerote FITC CD115 (AFS98) ebiosiene PE, APC CD34 (RAM34) ebiosiene efluor 660 CD16/32 (93) ebiosiene PE CD127 / IL 7Rα (A7R34) ebiosiene PERCP Cy5.5 S 1 / Ly6A/E (D7) ebiosiene PE Cy7 Kit / CD117 (ACK2) ebiosiene APC efluor 780 Mouse hemtopoieti linege oktil HO 1 (SPA 895, polylonl rit ) ebiosiene efluor 450 Assy Designs None, primry ntiody Polylonl rit serum Covne None, primry ntiody F( )2 Anti Rit IgG (seondry ntiody) ebiosiene FITC, seondry ntiody
Supplementary figure 1
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