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Assay vir Beta-galaktosidase-aktiwiteit in enkelselmikroskopie

Assay vir Beta-galaktosidase-aktiwiteit in enkelselmikroskopie


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Ek wil graag die aktiwiteit van $eta$-galaktosidase in lewende selle kan meet met eenvoudige optiese (miskien fluoressensie) mikroskopie. Ideaal gesproke wil ek 'n minimum van genetiese ingenieurswese doen, en hierdie toets gebruik met stamme wat ek reeds het (wat 'n WT-laktosestelsel het), d.w.s. 'n fluoresserende laktose-nabootsing sou ideaal wees. Daarbenewens sal dit lekker wees as die leeftyd van die fluoresserende neweproduk van $eta$-gal-aktiwiteit kort was, sodat ek 'n afname in $eta$-gal-aktiwiteit sowel as 'n toename kon opspoor. Bestaan ​​so 'n fluoresserende analoog? Ek hoop om te kyk na die oorskakeling van laktose na glukose benutting onder die loep, so ek wil hê dat laktose-gebruik selle lig en glukose-gebruik selle om donker te wees, of andersom. Dit hoef nie ten volle kwantitatief te wees nie - 'n kwalitatiewe sin is goed.


Ek het hierdie vraestel gevind.

Zhang GJ et al. (2009) In vivo optiese beelding van LacZ uitdrukking met behulp van lacZ transgeniese muise. Assay Drug Dev Technol.7:391-9. doi: 10.1089/adt.2009.0195.

Abstrak

beta-galaktosidase (beta-gal) (gekodeer deur die lacZ geen) is wyd gebruik as 'n transgeen verslaggewer ensiem. Die vermoë om lacZ-uitdrukking in lewende transgeniese diere te beeld sal die gebruik van hierdie verslaggewer verder uitbrei. Daar is gerapporteer dat 7-hidroksi-9H-(1,3-dichloor-9,9-dimetielakridien-2-oon)-beta-d-galaktopiranoside (DDAOG), 'n konjugaat van beta-galaktose en 7-hidroksie-9H -(1,3-dichloro-9,9-dimethylacridin-2-one), is nie net 'n chromogeniese lacZ substraat nie, maar dat die splitsingsproduk ver-rooi fluoressensie-eienskappe het wat deur in vivo beelding waargeneem kan word. In 'n poging om lacZ-uitdrukking in vivo nie-indringend te beeld, het ons fluoressensiebeelding toegepas op 'n G-proteïengekoppelde reseptor (GPR56), uitklop- (KO) muismodel, waarin die lacZ-geen in die GPR56-lokus ingevoer word. In vergelyking met wilde-tipe (WT) muise, het GPR56KO/LacZ muise drie- tot viervoudig hoër fluoressensie-intensiteit in die koparea 5 min na stert-aar inspuiting van DDAOG getoon. beta-Gal-kleuring in dele van die hele brein het sterk lacZ-uitdrukking in homosigote getoon, maar nie in WT-muise nie, in ooreenstemming met lacZ-aktiwiteit wat deur in vivo beelding opgespoor is. Die niere is ook gevisualiseer met fluoressensie beelding beide in vivo en ex vivo, in ooreenstemming met beta-gal kleuring bevindinge. Ons resultate toon dat fluoressensie beelding gebruik kan word vir in vivo intydse opsporing van lacZ aktiwiteit deur fluoressensie beelding in lacZ transgeniese muise. Hierdie tegnologie kan dus moontlik gebruik word om veranderinge van sekere endogene molekules en/of molekulêre weë in transgeniese diere nie-indringend te beeld.

Die werk in die koerant word aangehaal in 'n meer onlangse artikel wat beta-gal-gebaseerde chemiluminescerende beelding in muise rapporteer.

Dit is onduidelik of enige hiervan suksesvol sal wees op 'n enkele bakteriese selvlak.


Ek het dit as 'n antwoord geplaas omdat dit te lank was vir 'n opmerking. Oor die algemeen is jou eksperimentele benadering haalbaar, maar die duiwel is in die besonderhede.

Eerstens sal jou grootste uitdaging wees om toegang tot geskikte optika te kry. Die beeld van individuele eukariotiese selle is relatief maklik met enige diffraksiebeperkte optiese mikroskoop, want hul skaal is gewoonlik 10-20 $mu$m. Bakterieë is 'n baie kleiner selgrootte, en hulle kan (meestal) nie afgebeeld word deur diffraksiebeperkte tegnieke op 'n enkele selvlak te gebruik nie. Om 'n enkele sel te beeld vereis super-resolusie optika/tegnieke.

Tweedens, dit klink asof jy diauxic shift curves doen. Dit is dalk nie nodig om op 'n enkele selvlak te beeld nie, en jy kan dalk wegkom met die maak van goeie metings van selle in totaal met enige kamera-toegeruste mikroskoop, of selfs 'n optiese plaatleser, aangesien jy $eta$- genoem het. gal.

@Alan Boyd het 'n chemiluminescerende substraat vir $eta$-gal uitgewys wat goed kan werk as jy toegang het tot 'n optiese beeldvormer wat jou uitlesings van fotontellings/sekonde kan gee. Dit sal jou lesings gee van gemiddelde ensiematiese aktiwiteit (wat jy kan korreleer met uitdrukkingsvlak).

Jy beskryf ook dat selle helder/donker is, afhangende van die gebruikte substraat en dit sal moeiliker wees om te bereik. Daar is baie maniere wat ek voorstel om dit te doen, maar hulle verg almal 'n mate van genetiese manipulasie. Miskien is die maklikste ding om te doen om 'n uitdrukkingsplasmied te maak wat LacZ onder beheer van 'n IPTG-induseerbare promotor plaas. Hierdie plasmiede is baie algemeen en sal jou 'n maklike toegang gee tot die beeld van laktosegebruik.


Hier is 'n skakel na op die Invitrogen/Life Technologies-webblad waarin verskeie probes uiteengesit word om die aktiwiteit van glikosidases op te spoor, insluitend $eta$-galaktosidase. Dit sluit in DDAOG soos genoem deur @Alan Boyd, asook Fluorescein Digalactoside, Resorufin Galactoside, en Methylumbelliferyl Galactoside, onder andere. NB, dit is 'n Invitrogen/Life Technologies-webwerf, so al die reagense wat hulle noem, is dié wat deur die maatskappy verkoop word; daar mag ander wees wat nie hier genoem word nie. Hiervan is DDAOG baie stabiel, wat dit moontlik maak om die aanvang van $eta$-gal-aktiwiteit te meet, maar sal dalk nie werk om afnames in $eta$-gal-aktiwiteit te meet nie (sonder om die kleurstof te bleik). Resorufin Galactoside is sensitief en stabiel by fisiologiese pH, wat meting oor 'n lang tydperk moontlik maak. Fluoresceïen Digalaktosied is die mees sensitiewe toets, maar aangesien dit die verwydering van 2 galaktose-eenhede vereis vir volle fluoressensie, kan dit se aktiveringstyd stadig wees.

Vir my spesifieke gebruik (beperkte toegang tot amper-rooi frekwensies), dink ek dat Resorufin Galactoside of Fluorescein Digalactoside die beste keuses kan wees. Gegewe die koste van hierdie probes, kan ek egter beter daaraan toe wees om net 'n laboratorium te vind met 'n stam met gfp onder die lac-promotor in die chromosoom en hulle vir 'n monster te vra!


Protokolle om veroudering-geassosieerde beta-galaktosidase (SA-βgal) aktiwiteit op te spoor, 'n biomerker van verouderende selle in kultuur en in vivo

Normale selle kan permanent die vermoë verloor om te prolifereer wanneer hulle uitgedaag word deur potensieel onkogene stres, 'n proses wat sellulêre veroudering genoem word. Veroudering-geassosieerde beta-galaktosidase (SA-βgal) aktiwiteit, waarneembaar by pH 6.0, laat die identifikasie van verouderde selle in kultuur en soogdierweefsels toe. Hier beskryf ons eers 'n sitochemiese protokol wat geskik is vir die histochemiese opsporing van individuele senesente selle in beide kultuur en weefselbiopsies. Die tweede metode is gebaseer op die alkalisering van lisosome, gevolg deur die gebruik van 5-dodecanoylaminofluorescein di-β-D-galaktopiranoside (C)12FDG), 'n fluorogeniese substraat vir βgal-aktiwiteit. Die sitochemiese metode neem ongeveer 30 minute om uit te voer, en 'n paar uur tot 'n dag om te ontwikkel en te score. Die fluoressensiemetodes neem tussen 4 en 8 uur om uit te voer en kan in 'n enkele dag aangeteken word. Die sitochemiese metode is van toepassing op weefselafdelings en vereis eenvoudige reagense en toerusting. Die fluoressensie-gebaseerde metodes het die voordele dat dit meer kwantitatief en sensitief is.


Metodes om biomerkers van sellulêre veroudering op te spoor: die veroudering-geassosieerde beta-galaktosidase-toets

Die meeste normale menslike selle ondergaan sellulêre veroudering nadat hulle 'n vaste aantal seldelings opgedoen het, of word uitgedaag deur 'n verskeidenheid potensieel onkogene stimuli, in kultuur en heel waarskynlik in vivo. Sellulêre veroudering word gekenmerk deur 'n onomkeerbare groeistop en sekere veranderde funksies. Bejaarde selle in kultuur word geïdentifiseer deur hul onvermoë om DNS-sintese te ondergaan, 'n eienskap wat ook deur rustige selle gedeel word. Etlike jare gelede het ons 'n biomerker beskryf wat geassosieer word met die verouderingsfenotipe, 'n verouderingsgeassosieerde beta-galaktosidase (SA-beta-gal), wat opgespoor word deur histochemiese kleuring van selle met behulp van die kunsmatige substraat X-gal. Die teenwoordigheid van die SA-beta-gal biomerker is onafhanklik van DNA-sintese en onderskei gewoonlik verouderde selle van russelle. Die metode om SA-beta-gal op te spoor is 'n gerieflike, enkelsel-gebaseerde toets, wat verouderde selle selfs in heterogene selpopulasies en verouderende weefsels kan identifiseer, soos velbiopsies van ouer individue. Omdat dit maklik is om op te spoor, is SA-beta-gal tans 'n wyd gebruikte biomerker van veroudering. Hier beskryf ons 'n metode om SA-beta-gal in detail op te spoor, insluitend 'n paar onlangse wysigings.


Enkelsel epigenetiese visualiseringstoets

Karakterisering van die epigenetiese status van individuele selle bly 'n uitdaging. Huidige volgordebepalingsbenaderings het beperkte dekking, en dit is moeilik om 'n epigenetiese status aan die transkripsietoestand van individuele geenallele in dieselfde sel toe te ken. Om hierdie beperkings aan te spreek, is 'n geteikende mikroskopie-gebaseerde epigenetiese visualiseringstoets (EVA) ontwikkel vir opsporing en kwantifisering van epigenetiese merke by gene van belang in enkelselle. Die toets is gebaseer op 'n in situ biochemiese reaksie tussen 'n teenliggaam-gekonjugeerde alkaliese fosfatase gebind aan die epigenetiese merk van belang, en 'n 5'-gefosforileerde fluorofoor-gemerkte DNA-oligo wat deur geenspesifieke oligonukleotiede aan 'n teikengeen vasgemaak is. Wanneer die epigenetiese merk by die geen teenwoordig is, beskerm fosfaatgroepverwydering deur die fosfatase die oligo teen λ-eksonuklease-aktiwiteit wat 'n kwantitatiewe fluoresserende uitlees verskaf. Ons het EVA toegepas om 5-metielsitosien (5mC) en H3K9Ac vlakke by verskillende gene en die MIV-1 provirus in menslike sellyne te meet. Om epigenetiese merke aan geentranskripsie te koppel, is EVA gekombineer met RNA-FISH. Hoër 5mC-vlakke by die stilgemaakte in vergelyking met getranskribeerde XIST-geenallele in vroulike somatiese selle het hierdie benadering bekragtig en gedemonstreer dat EVA gebruik kan word om epigenetiese merke met die transkripsiestatus van individuele geenallele in verband te bring.

© The Author(s) 2021. Gepubliseer deur Oxford University Press namens Nucleic Acid Research.

Syfers

Epigenetiese merkvisualisering by 'n ...

Epigenetiese merkvisualisering by 'n geen van belang. Alkaliese fosfatase (AP) word gewerf ...

RNA VIS—EVA-analise van rDNA...

RNA VIS—EVA analise van rDNA lokus. ( A ) rDNA-transkripsie-eenheid en...

Afgedrukte XIST-geen. ( 'n …

Afgedrukte XIST-geen. ( A ) Menslike XIST geen lokus en sonde ontwerp.…

Enkelkopie EGR1 geen. (…

Enkelkopie EGR1 geen. ( A ) Mens EGR1 geen lokus en sonde …

MIV-provirus in latent besmette...

MIV-provirus in latent besmette selle. ( A ) Skematiese oorsig van die...

Histoon-modifikasie H3K9Ac EVA-analise ...

Histoonmodifikasie H3K9Ac EVA-analise van rDNA. ( A ) H3K9Ac-EVA-toets was...


SA-β-galaktosidase-gebaseerde siftingstoets vir die identifisering van senoterapeutiese middels

Sellulêre veroudering is die sleutelfaktor in die ontwikkeling van chroniese ouderdomverwante patologieë. Identifikasie van terapeutiese middels wat senesente selle teiken toon belofte vir die verlenging van gesonde veroudering. Hier bied ons 'n nuwe toets aan om te skerm vir die identifikasie van senoterapeutika gebaseer op meting van veroudering geassosieer β-Galactosidase aktiwiteit in enkelselle.

Ons beskryf hier vir die eerste keer 'n metode wat 'n hoë-deurset dwelm sifting moontlik maak vir senoterapeutiese verbindings wat getoon is voordelig te wees in veroudering-verwante siektes. Trouens, verskeie kliniese proewe met dwelms wat in hierdie essensie getoets is, is tans aan die gang. Die hoofvoordeel van hierdie tegniek is om te onderskei tussen twee tipes senoterapeutiese middels, senolitika en senomorfies, deur die goed gevestigde opsporing van SA-beta-Galaktosidase in senesente selle te gebruik.

Hierdie tegniek is 'n belangrike siftingsinstrument vir nuwe senoterapeutiese verbindings. Trouens, verskeie verbindings wat met behulp van hierdie skerm geïdentifiseer is, word in verskeie hoëprofielpublikasies vertoon, en is tans in kliniese proefneming. Hierdie tegniek is uiters veelsydig, en kan aangepas word vir verskeie hoë-deurset skerms.

Trouens, dit is moontlik om skerms te vermenigvuldig en te skerm vir verbindings wat veroudering onderdruk deur die aktivering of inhibisie van een van verskeie weë. Maal elke embrio in stukke van een millimeter en pyp dit verskeie kere op en af. Voeg dan 10 milliliter groeimedium by die weefsels van elke embrio.

Plaat hulle in 'n 10 sentimeter kultuurskottel, en inkubeer by 37 grade Celsius in 3% suurstof en 5% koolstofdioksied vir twee tot drie dae. En inkubeer die res van elke embrio met 0,25% tripsien EDTA mengsel vir 10 minute. Om selle te tripsiniseer, verwyder eers die groeimedium versigtig en was die selle twee keer met 10 milliliter 1X PBS.

Voeg dan twee milliliter van 'n 025 tripsien EDTA oplossing by die selle, en inkubeer hulle by 37 grade Celsius vir twee tot drie minute. Inspekteer dan die selle onder 'n mikroskoop om seker te maak dat die selle van die oppervlak losgemaak is. Voeg dieselfde hoeveelheid groeimedium by om die tripsienvertering te beëindig, en dra die selle oor na 'n koniese buis.

Sentrifugeer dan die selle by 200 keer G vir drie minute. Gooi die supernatant weg en voeg vars groeimedium by om die selle te hersuspendeer. Tel dan die selle en saai hulle in nuwe plate teen die geprojekteerde seldigtheid.

Vir nie-verouderende subkultivering, verdeel eers samevloeiende selle teen 'n een-tot-vier-verhouding. Inkubeer dan selle by 37 grade Celsius in 3% suurstof en 5% koolstofdioksied om uit te brei vir nog 'n deurgang. Vervolgens, om selveroudering te veroorsaak, split saad samevloeiende selle uit gang een in 'n verhouding van een tot vier en inkubeer hulle by 37 grade Celsius in 20% suurstof en 5% koolstofdioksied omgewing vir drie dae.

Om sellulêre veroudering te monitor, met 'n gevorderde Coulter Cell Counter-stelsel, meet die geleidelike toename in seldeursnee en selvolume tydens elke tripsiniseringstap. Om beta-Gal-siftingstoets te begin, saai 5 000 senescent selle of 3 000 nie-senescent selle per put in 96 put plate. Voeg dan 100 mikroliter groeimedium by elke put en inkubeer selle by 37 grade Celsius en 20% suurstof en 5% koolstofdioksied vir ses uur.

Voeg dan geneesmiddelverdunnings by MEF-selle en inkubeer die selle by 37 grade Celsius in 20% suurstof en 5% koolstofdioksied vir 24 tot 48 uur. Na inkubasie, verwyder die geneesmiddeloplossing en was selle met 100 mikroliter 1X PBS. Om lisosomale alkalinisering te bewerkstellig, behandel selle vooraf met 90 mikroliter bafilomycin A1 oplossing, verdun in vars selkultuurmedium teen 'n finale konsentrasie van 100 nanomolêr.

Inkubeer dan selle by 37 grade Celsius in 20% suurstof en 5% koolstofdioksied omgewing vir 'n uur. Vir fluoressensie-analise van SA-beta-Gal-aktiwiteit, maak 'n vars werkende oplossing van C12FDG verdun in groeimedium, teen 'n finale konsentrasie van 100 mikromolêr. Voeg dan 10 mikroliter van die werkende oplossing by die kultuurmedium teen 'n finale konsentrasie van 10 mikromolêr, en inkubeer selle by 37 grade Celsius in 20% suurstof en 5% koolstofdioksied vir twee uur.

Was selle met 100 mikroliter 1X PBS. Voeg dan twee mikroliter van 'n 100 mikrogram-per-milliliter Hoechst 33342-kleurstof by teen 'n finale konsentrasie van twee mikrogram per milliliter. Inkubeer die selle by 37 grade Celsius, 20% suurstof en 5% koolstofdioksied vir 20 minute.

Na inkubasie, verwyder die media, voeg 100 mikroliter vars groeimedium by en gaan voort met beeldvorming. In hierdie studie het 'n hoë-inhoud fluoresserende beeld verkryging en analise platform die identifikasie van senolitiese middels, soos 17-DMAG, moontlik gemaak wat die algehele SA-beta-Gal aktiwiteite in muriene embrioniese fibroblast selkulture wat senesente selle in passasie vyf bevat, verminder het. Die sagteware-gegenereerde kwantitatiewe ontledings van mengsels van verouderende en nie-verouderende selle maak voorsiening vir die duidelike demonstrasie van verouderende sel-spesifieke effekte, en die uitskakeling van agtergrond beta-galaktosidase aktiwiteit deur bafilomycin A. Maak seker dat alle nodige selkontroles ingesluit is, veral onbehandelde senesente selle en senescent selle behandel met 'n positiewe kontrole.

Hierdie opstel sluit in werk met primêre selle onder oksidatiewe stres, wat kan lei tot variasie wat gemonitor moet word. Hierdie prosedure is die eerste stap in die opsporing van senoterapeutiese middels. Bykomende sellewensvatbaarheid en selverouderende opstelle moet uitgevoer word om die siftingsresultate te bevestig.

Verskeie nuwe senolitiese middels is opgespoor met behulp van hierdie opstel, insluitend die eerste senolitiese dwelm kombinasie dasatinib quercetin, hitteskokproteïen 90 inhibeerders, Bcl-2 familie inhibeerders, en die polifenol fisetin.


Assay vir Beta-galaktosidase-aktiwiteit in enkelselmikroskopie - Biologie

'n Omvattende oorsig van toetse vir selproliferasie en sellewensvatbaarheid, saam met die resultate van 'n Labome-opname van formele publikasies.

Daar is tans baie metodes en stelle beskikbaar wat gebruik word om talle aspekte van selproliferasie, mitochondriale funksie en, indirek, sellewensvatbaarheid te meet. Oor-analise van hierdie toetse en verkeerde interpretasie van die inligting wat hulle verskaf, word egter meer gereeld in die gepubliseerde literatuur. In 'n selproliferasietoets behoort die uitset vir jou 'n direkte en akkurate meting te gee van die aantal aktief-delende selle in 'n populasie, of dit nou selle in kultuur of weefsel is. Daarteenoor is 'n sellewensvatbaarheidstoets ontwerp om 'n aanduiding te gee van die aantal "gesonde" selle binne 'n populasie, gereeld deur spesifieke aanwysers van metabolies aktiewe selle te assesseer, wat dikwels verband hou met mitochondriale funksie. In teenstelling met 'n proliferasietoets, onderskei hierdie metings nie tussen rustige/verouderende en aktief-delende selle nie. Beta-galaktosidase-aktiwiteitstoets of -kleuring kan verouderde selle aandui [1]. Toetse vir die selsiklusanalise en seldood word elders bespreek.

Etiketvrye benaderings, soos xCELLigence RTCA SP/DP Real Time Cell Analyzer van ACEA Biosciences, kan die toename in selgetalle meet, en dus die seladhesie, proliferasie, loslating/dood kwantifiseer [2, 3].

Die tradisionele metode vir die bepaling van selproliferasie is om DNS-sintese te meet deur die inkorporering van 'n gemerkte DNS-analoog of voorloper (5-broom-2'-deoksiuridien (BrdU), 'n analoog van pirimidien wat dit in nuwe DNS in die plek geïnkorporeer word, te bepaal. van timidien, of ΑH]-timidien) in die genomiese DNA van selle tydens S-fase van die selsiklus. Maun HR et al het byvoorbeeld die proliferasie van brongiale gladdespierselle met 3H-timidine inkorporasie beoordeel [4]. Vir selle in kultuur is die mees algemene metode om BrdU-inkorporering deur kolorimetriese ELISA te assesseer (Figuur 1). Soortgelyke metodes kan gebruik word om prolifererende selle te assesseer in vivo deur pols-etikettering van weefsels met BrdU voor oes, gevolg deur die assessering van BrdU deur ELISA of immunohistochemiese kleuring. Dit kan ook beoordeel word deur vloeisitometrie 'n voorbeeldspoor van BrdU vs. 7-AAD word in Figuur 2 getoon.

'n Nuwer benadering is om die alkyn-bevattende timidien-analoog EdU (5-etyniel-2'-deoksiuridien) in DNA in te sluit en die geïnkorporeerde EdU op te spoor deur 'n klikreaksie - 'n koper-gekataliseerde asied-alkyn sikloadisie [5] - in die geval van etikettering in lewende diere, kan EdU óf ingespuit óf in die drinkwater gemeng word [6]. Ouadah Y et al het daagliks in muise ingespuit, ip, BrdU en Edu en die gemerkte selle opgespoor deur 'n anti-BrdU-teenliggaam (Abcam ab6326) en Click-iT EdU-stelle van Thermo Fisher op die krio-afdelings om die aktivering van pulmonêre neuro-endokriene selle te ondersoek [ 7]. 'n Marconi et al.-pulsgejaagde chondrogenese in embrioniese en volwasse skaats, Leucoraja erinacea deur EdU-etikettering en opsporing met Click-iT EdU Cell Proliferation Kit van Thermo Fisher [8]. Prolifererende EdU+-selle kan ook deur vloeisitometrie opgespoor word met die Click-iT Plus EdU-vloeisitometrie-toetsstel van Thermo Fisher [9].

BrdU self kan die selfisiologie beïnvloed. Dit kan transkripsiefaktor- en chemiese-geïnduseerde herprogrammering bevorder [10] en kan ook DNS-struktuur verander [11].

SimProteïenTop drie verskaffers
H3-3AH3.3 histoon ASelseintegnologie 9733 (107), Abcam ab14955 (31), aktiewe motief 39763 (18)
MCM2minichromosoom onderhoudskompleks komponent 2Selseintegnologie 3619 (7), BD Biowetenskappe 610700 (5), Abcam ab108935 (2)
MKI67merker van verspreiding Ki-67Invitrogen MA5-14520 (771), Dako M7240 (271), Abcam ab16667 (193)
PCNAprolifererende selkernantigeenSanta Cruz Biotechnology sc-56 (190), Invitrogen MA5-11358 (181), Abcam ab29 (71)

In afdelings van vaste diereweefsels en selpopulasies word proliferasie gewoonlik geassesseer deur immunokleuring vir spesifieke proliferatiewe merkers, waarvan sommige hier beskryf word. Ki-67 is 'n kernproteïen wat verband hou met selproliferasie en ribosomale RNA-transkripsie [12] en algemeen gebruik [13]. Tradisionele Ki67-teenliggaampies is beperk deurdat hulle slegs gebruik kan word om bevrore, en nie paraffien-ingebedde dele, te kleur nie. Nuwe MIB-1-teenliggaampies, gerig teen 'n ander epitoop van Ki67, kan egter ook gebruik word om formalien en paraffien-gefixeerde snitte te kleur, verhoog die toediening van Ki67 as 'n proliferatiewe merker. Ouadah Y et al het muislong-kryoseksies met Ki-67-teenliggaampies (Thermo Fisher 41-5698-82 en 50-5698-82) gekleur om beserings-geïnduseerde neuro-endokriene selproliferasie te ondersoek [7]. Nam S et al het Ki-67 kleuring en vloeisitometrie gebruik om fraksie van selle van 3D hidrogelkultuur in G0 en G1 fases van die selsiklus te skat [14]. Hu CK et al, aan die ander kant, het selle gekleur tydens die G2/M-oorgang van die selsiklus in killifish-embrio's met Hoechst en teenliggaampies teen fosfo-Histone H3 (Ser10) om selproliferasie te bepaal [15].

Prolifererende selkernantigeen (PCNA) is nog 'n algemeen gebruikte merker van selproliferasie. Dit bespoedig DNA-sintese met DNA-polimerase-δ deur die genoom te omsingel wat replikasie vergemaklik deur die polimerase aan die DNA te hou. Dit word dus tydens DNA-sintese in die kern uitgedruk en kan as sodanig as 'n merker van selproliferasie gebruik word. Dit speel ook 'n belangrike rol in DNA-herstel. Ander prolifererende merkers sluit in MCM2 [16].

Alle toetse wat DNS-sintese direk of indirek meet, is intrinsiek sensitief vir die stadium van die selsiklus. Afhangende van die uitkoms van die toets, kan dit dus nodig wees om die selle te sinchroniseer, hetsy deur serumonttrekking wat selle in G1 ophoop (wat ook lewensvatbaarheid kan beïnvloed) of DNA-sintese chemies te inhibeer, wat selle in S-fase blokkeer, met timidien , sitosien arabinosied, hidroksiureum of aminopterien. Boonop kan selproliferasie ook gemeet word deur monitering van seldeling deur byvoorbeeld Cell Proliferation Dye eFluor™ 670 van Thermo Fisher te gebruik [17].

Selgebaseerde toetse om lewensvatbaarheid te meet kan hoofsaaklik in drie kategorieë verdeel word: dié wat die verlies aan membraanintegriteit ontgin, dié wat metaboliese merkers direk meet, en dié wat metaboliese aktiwiteit assesseer. Ander vorme van opsporing bestaan. Kristalvioletkleuring kan die aanhegting van selle kontroleer en dus die lewensvatbaarheid van aanhechtende selle meet [18]. Lee YR et al het byvoorbeeld kristalvioletkleuring gebruik om die effekte van PTEN K342/K344R-mutant op die verspreiding van PC3-selle te bepaal [19]. Vasan N et al het vaste MCF10A-selle met kristalviolet gekleur om selproliferasie te meet [20].

  • MTT: MTT (3-(4,5-dimetieltiasool-2-yl)-2,5-difeniel-tetrazoliumbromied) is 'n tetrazoliumsout wat deur beide mitochondriale en ekstra-mitochondriale dehidrogenases verminder word om onoplosbare blou formazan-kristalle te vorm, wat beteken 'n solubiliseringstap is nodig voordat die toets gelees kan word [21]. Boonop word selle nie-lewensvatbaar tydens hierdie toets, wat beteken dat herhaalde of komplementêre toetse nie op dieselfde plaat selle uitgevoer kan word nie.
  • MTS/XTT: MTS (3-(4,5-dimetieltiasool-2-yl)-5-(3-karboksimetoksifeniel)-2-(4-sulfofeniel)-2H-tetrazolium) en XTT (2,3-bis-( 2-metoksi-4-nitro-5-sulfofeniel)-2H-tetrazolium-5-karboksanilied) substrate is soortgelyk aan MTT. Een voordeel is egter dat die reaksies intrasellulêr uitgevoer word in die teenwoordigheid van die intermediêre elektronaanvaarder fenasienmetosulfaat (PMS), wat hul sensitiwiteit verhoog. Daarbenewens is die gereduseerde formazan-produk oplosbaar en word vrygestel na die kultuurmedia, wat die behoefte aan die ekstra oplosbaarheidstap wat met MTT benodig word, verwyder. Daar is egter gerapporteer dat fenolrooi in selkultuurmedia, vetsure en serumalbumien data verkry van MTS-, XTT- en WST-toetse oor lang inkubasieperiodes verdraai [22]. Promega RealTime Glo MT Sellewensvatbaarheidstoets ondersoek die reduktiewe omgewing binne selle om 'n chemikalie tot 'n geskikte substraat vir NanoLuc-luciferase te reduseer. Reynders M et al het byvoorbeeld selsitotoksisiteit en proliferasie gemeet met 'n MTS-toets [23].
  • Alamar Blue: Alamar Blue is 'n resazurin-verbinding wat in lewensvatbare selle gereduseer word tot resorufien en dihidroresorufien. Dit kan lewende selle binnedring, vereis dus nie sellise nie, en is stabiel in kultuurmedia. Hierdie toets het die bykomende voordeel dat dit in beide fluorimetriese en kolorimetriese plaatlesers gemeet kan word. Silva MC et al het Thermo Fisher Alamar Blue-reagens gebruik om die sellewensvatbaarheid van geïnduseerde pluripotente sel-afgeleide neurale stamvaderselle te meet tydens behandeling van tau-proteïenafbreker QC-01-175 [24]. Dominy SS et al het die lewensvatbaarheid van menslike neuroblastoom SH-SY5Y-selle gemeet met Porphyromonas gingivalis met of sonder gingipain-inhibeerders of antibiotika met behulp van Thermo Fisher Alamar Blue-reagens [25].
  • WST: Wateroplosbare tetrazoliumsoute (WSTs) is sel-ondeurdringbare tetrazolium kleurstowwe wat ekstrasellulêr verminder word via plasmamembraan elektronvervoer [26], en gekombineer met die elektronontvanger PMS om wateroplosbare formazan kleurstowwe te genereer. Noda S et al, byvoorbeeld, het die lewensvatbaarheid van primêre tandpulp-stamselle ondersoek met Cell Counting Kit-8 (wat WST-8-oplossing bevat, ook genoem CCK-8-toets), van Dojindo Laboratories [27]. L Zhao et al het dieselfde stel gebruik om tumorselproliferasie in kultuur te meet [28]. Luther A et al het 'n soortgelyke WST-8-stel gebruik, maar van MilliporeSigma, om die sitotoksisiteit van voornemende antibiotika op HeLa- en HEP ​​G2-selle te meet [29].

Daar is talle beskikbare toetse wat ATP-vlakke meet as 'n uitset van algehele selgesondheid. Wanneer selle begin om apoptose te ondergaan of membraanintegriteit verloor, raak ATP-voorraad uitgeput deur die aktiwiteit van ATPases wat gelyktydig enige nuwe ATP-sintese voorkom. Dit lei tot 'n vinnige uitputting van intrasellulêre ATP-vlakke. Luminescerende ATP-toetse (soos Promega se CellTiter-Glo) funksioneer deur selle te lys om ATP-store vry te stel, terwyl ATPases gelyktydig geïnhibeer word. Luciferase kataliseer die oksidasie van luciferin na oxyluciferin in die teenwoordigheid van magnesium en ATP, wat lei tot 'n luminescerende sein wat direk korreleer met die intrasellulêre ATP-konsentrasie [30, 31].

Daar is 'n aantal besluite om te neem wanneer die geskikte metaboliese toets vir jou behoeftes gekies word. Elkeen van die substrate wat hierbo gelys is, en verwante wat nie hier gedek word nie, het hul duidelike voordele en nadele wanneer dit direk vergelyk word. Toetssensitiwiteit, geraas-tot-sein-verhouding, gebruiksgemak en reagensstabiliteit is almal faktore wat in ag geneem moet word. 'n Bykomende belangrike oorweging met metaboliese toetse is dat die vermindering van hierdie substrate beïnvloed word deur veranderinge in intrasellulêre metaboliese aktiwiteit wat geen direkte uitwerking op algehele sellewensvatbaarheid het nie, daarom sal die vraag wat jy probeer beantwoord 'n sleutelrol speel in die keuse van die toepaslike toetse.

  • LDH: Laktaatdehidrogenase is 'n alomteenwoordige, stabiele sitoplasmiese ensiem wat laktaat omskakel na piruvaat. As die selmembraan beskadig is, word LDH, en dus, sy ensiematiese aktiwiteit uit selle vrygestel en kan in selkultuurmedia opgespoor word. Tydens die omskakeling van laktaat na piruvaat, word NAD+ na NADH/H+ verminder. LDH-gebaseerde lewensvatbaarheidstoetse kapitaliseer op die vorming van die vrye waterstofioon deur die oordrag van H+ vanaf NADH/H+ na die tetrazoliumsout INT (2-(4-jodofeniel)-3-(4-nitrofeniel)-5-fenieltetrazoliumchloried te kataliseer ), verminder dit na 'n rooi formazan-kleurstof [32], of omskakeling van resazurin na die fluoresserende vorm resorufien in Promega CytoTox-ONE-toets [33]. Persson EK et al het Pierce LDH-sitotoksisiteit-toetsstel gebruik om nekrotiese seldood as gevolg van Charcot-Leyden-kristalle te meet [34].
  • Trypanblou: Kleuring van 'n selsuspensie met tripaanblou is een van die oudste en eenvoudigste lewensvatbaarheidtoetse [35, 36]. In 'n gesonde, lewensvatbare sel sal die ongeskonde membraan verhoed dat tripaanblou selle binnedring. In dooie of sterwende selle sal tripaanblou die sel binnegaan en dit blou vlek. Hierdie metode is tradisioneel met die hand gekwantifiseer deur mikroskope en hemositometers te gebruik, wat dit baie arbeidsintensief maak. Die onlangse beskikbaarheid van bekostigbare geoutomatiseerde seltellers, byvoorbeeld Bio-Rad TC20 outomatiese selteller [36] of Vi-CELL™ outomatiese sellewensvatbaarheidontleder en teller van Beckman Coulter [37], maak hierdie toets minder tydrowend en meer akkuraat as voorheen. Ander soortgelyke, maar fluoresserende kleurstowwe kan ook gebruik word. Byvoorbeeld, Aizarani N et al het lewensvatbare selle geïdentifiseer met 'n sel sorteerder deur Zombie Green van BioLegend [38].
  • Calcein-AM: Calcein-acetoxymethylester is 'n nie-fluoresserende kleurstof wat gebruik word in beide sel lewensvatbaarheid en apoptose toetse, byvoorbeeld, vir die opsporing van die sitotoksisiteit van Abeta peptied en sy oligomere [39]. Dit is lipofiel, wat maklik deur die selmembraan moontlik maak. Sodra dit binne die sel is, klief intrasellulêre esterase die esterbindings van die asetometoksiegroep, wat lei tot die vorming van 'n fluoresserende anioniese en hidrofiele kalseïenkleurstof, wat in die sel vasgevang word. Nie-lewensvatbare selle bevat nie aktiewe esterases nie, wat dit moontlik maak om hierdie toets as 'n maatstaf van lewensvatbaarheid te gebruik. Cu 2+ , Co 2+ , Fe 3+ , Mn 2+ en Ni 2+ blus die fluoresserende sein vanaf kalseïen by fisiologiese pH, wat beteken dat sorg gedra moet word om die toepaslike selkultuurmedia te kies.
  • Propidium Jodide/7-AAD: Hierdie interkalerende middels word gereeld gebruik om die selsiklus te bestudeer soos hierbo bespreek. Aangesien hulle egter membraan-ondeurdringbaar is, word hulle uitgesluit van lewensvatbare selle. Dit beteken dat die fluoressensiesein wat deur PI of 7-AAD in nie-lewensvatbare selle uitgestraal word, gemeet kan word deur fluoressensiemikroskopie [40] of FACS-analise [41-43]. Byvoorbeeld, Nortley R et al het die perisiet-seldood geëvalueer met 7.5 uM propidiumjodied [44]. Capello M et al het IncuCyte™ Cytotox Green Reagent van Essen Bioscience gebruik om die seldood onder gekweekte kankerselle te meet [45].
  • Selondeurdringbare DNA-bindende kleurstowwe soos DRAQ7 van Abcam [46] of SYTOX van Thermo Fisher. Hierdie kleurstowwe gaan selle binne deur gekompromitteerde selmembrane en vertoon sterk fluoressensie by binding met DNA. Byvoorbeeld, Samir P et al het seldood in gekweekte beenmurg-afgeleide makrofage in reële tyd gemonitor met SYTOX Green-kleuring onder 'n tweekleur IncuCyte Zoom-broeikasbeeldstelsel van Essen Biosciences [47].

This section is provided by Labome to help guide researchers to identify most suited cell proliferation and cell viability assay kits. Labome surveys formal publications. Table 2 lists the major suppliers for reagents/kits used in the cell-based assays. Some of the applications are enumerated here. Zeng Q et al measured breast cancer cell growth with an MTT cell proliferation kit from Roche [48]. Nam S et al measured cell proliferation in 3D-hydrogel with EdU from Thermo Fisher Scientific [14]. Ombrato L et al assessed the in vitro proliferation of MMTV–PyMT actin–GFP cells in 2D co-culture with MACS-sorted EPCAM+ and Ly6G+ mouse cells using Click-iT Plus EdU Flow Cytometry Assay Kit from Thermo Fisher in a flow cytometer [49]. Silvestre-Roig C et al assayed the cell viability of smooth muscle cells incubated with isolated NETs with propidium iodide, Vybrant MTT cell proliferation assay from Thermo Fisher, and for live imaging, calcein AM from Thermo Fisher [50]. Genet G et al measured the proliferation of HUVEC cells in response to VEGF-A with the xCELLigence RTCA DP analyzer from ACEA Biosciences [3]. Herb M et al assessed the viability of macrophages in culture with the CyQUANT Direct Cell Proliferation Assay from Thermo Fisher Scientific [51].

supplierkitmethodssteekproefverwysings
AbcamDRAQ7cytometry or flow cytometry [46]
Abcamcalcein AMflow cytometry [52]
ACEA BiosciencesxCELLigence RTCA DP analyzerlabel-free [3]
BD PharmingenBrdU Flow Kitflow cytometry [53]
BioLegendcarboxyfluorescein succinimidyl esterflow cytometry [54]
BioLegendTag-it-violetflow cytometry [41]
BioLegendZombie Aquaflow cytometry, immunocytochemistry [55-57]
BioLegendZombie GreenFACS [38]
Dojindo LaboratoriesCell Counting Kit-8 Greenkolorimetrie [27]
Essen BioscienceIncuCyte Cytotox Green [45]
MilliporeSigmaBrdUimmunohistochemistry [7]
MilliporeSigmaCell Counting Kit-8 [29]
MilliporeSigmaMTT [48, 58]
PromegaCellTiter 96 AQueouscell imaging [41, 45, 59, 60]
PromegaCellTiter-Gloluminescent assay [46, 61]
PromegaCellTiter-Glo 3Dluminescent assay [62]
PromegaCytoTox 96 [63, 64]
PromegaCytoTox-ONE [33]
PromegaRealTime-Glo MT [65]
RocheWST-1 [66]
Thermo FisherAlamar Bluebeelding [24, 25]
Thermo Fishercalcein AMbeelding [50]
Thermo FisherCell Proliferation Dye e670flow cytometry [17]
Thermo FisherCellTrace Violet Cell Proliferationflow cytometry [53]
Thermo FisherClick-iT Eduimmunochemistry, flow cytometry [7, 67]
Thermo FisherCyQUANT Direct Cell Proliferation Assaycell imaging [68, 69]
Thermo FisherLIVE/DEAD Fixable Blue Dead Cell Stain Kitflow cytometry [42]
Thermo FisherSYTOXcell imaging [47, 62]
Thermo FisherVybrant MTTkolorimetrie [70]

This article is derived from an earlier version of an article authored by Dr. Laura Cobb "Cell-Based Assays: the Cell Cycle, Cell Proliferation and Cell Death", written in February 2013.


Flow Cytometric Single-Cell Analysis for Quantitative in Vivo Detection of Protein-Protein Interactions via Relative Reporter Protein Expression Measurement

Cell-based two-hybrid assays have been key players in identifying pairwise interactions, yet quantitative measurement of protein-protein interactions in vivo remains challenging. Here, we show that by using relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, quantitative analysis of protein interactions in a bacterial adenylate cyclase two-hybrid (BACTH) system can be achieved. A multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the β-galactosidase (β-gal) reporter protein upon dual immunofluorescence staining. Single-cell analysis revealed that there exists bistability in the BACTH system and the RRPE is an intrinsic characteristic associated with the binding strength between the two interacting proteins. The RRPE-BACTH method provides an efficient tool to confirm interacting pairs of proteins, investigate determinant residues in protein-protein interaction, and compare interaction strength of different pairs.


Trypan Blue Cell Counting

Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live (viable) cells do not take up the blue dye, whereas dead (non-viable) cells do. Cell viability can be calculated using the ratio of total live/total cells (live and dead). Staining also facilitates the visualization of overall cell morphology.

NOTE: Trypan Blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark due to the presence of serum in the matrix, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting.

Figuur 6. Cell Counting Using a Hemocytometer and Trypan Blue


Kinetic Analysis of ß-Galactosidase Activity using PowerWave™ HT Microplate Spectrophotometer and Gen5™ Data Analysis Software

The determination of the enzyme kinetic parameters for newly discovered proteins is an important procedure in cellular and molecular biology. Here we describe the use of kinetic reading for the analysis of the bacterial enzyme, ß-galactosidase, using o-nitrophenol-ß-D-galactoside (ONPG) as the substrate.

Inleiding

Most biological processes require an enzyme to act as a catalyst in order for the reaction to take place. With the exception of catalytic RNA, all enzymes are proteins. Most importantly, enzymes are highly effective in catalyzing diverse chemical reactions in a regulated, selective, and specific manner. With a few exceptions, most reactions catalyzed by enzymes can be described satisfactorily by the Michaelis-Menten Equation (Eq. 1),

where v = reaction rate, [S] = substrate concentration, Vmaks = maximal velocity, and Km is the Michaelis constant. Each enzyme has physical characteristics with regard to substrate specificity, reaction velocity or required cofactors that affect the Michaelis-Menten constants. The determination of these constants involves the performance of kinetic enzymatic activity measurements. Here we utilize an absorbance-based assay for ß-galactosidase enzyme activity to demonstrate the capabilities of the PowerWave HT Microplate Spectrophotometer in conjunction with Gen5 Data Analysis Software to perform routine analysis of enzyme kinetics in microplates.

The enzymatic product of the LacZ gene, ß-galactosidase, catalyses the hydrolysis of ß-D-galactosides, such as lactose, into their component sugars by hydrolysis of the terminal nonreducing ß-D-galactose residues (Figure 1).

Fortunately the substrate specificity of the enzyme is such that a variety of different substrates, each with a ß-D-galactopyranoside moiety, can be acted upon. Investigators have taken advantage of this by synthesizing compounds which when hydrolyzed by ß-galactosidase result in a colored product. One such compound is o-nitrophenol-ß-D-galactoside (ONPG), which when hydrolyzed forms galactose and o-nitrophenol (Figure 1).

The compound ONP absorbs light at 420 nm whereas the precursor molecule ONPG does not. Therefore, the increase in light absorbance at 420 nm can be used to monitor ß-galactosidase when ONPG is used as a substrate.

Other compounds that possess a conformation similar to ONPG or lactose (i.e. have a ß-D-galactose moiety), can be used in combination with ß-galactosidase. There are several compounds, such as 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal), that form colored insoluble products after hydrolysis. These compounds are quite useful in screening for bacterial plasmid recombinants (1). Bacterial cells containing the LacZ geen (Lac + ) would hydrolyze X-gal and turn blue, while Lac- cells, unable to hydrolyze the compound would remain white. Alternatively, compounds that cannot be hydrolyzed but retain a similar structure would be expected to act as an inhibitor. Phenylethyl ß-D-thiogalactopyranoside (PETG) has a thiol group substituted for the hydroxyl linkage present in compounds that ß-galactosidase normally hydrolyzes (Figure 2). PETG when present in a reaction would be expected to occupy the enzyme and thus inhibit its action on hydrolyzable substrates such as ONPG.

Materiale en Metodes

o-Nitrophenyl ß-D-galactopyranoside (ONPG), catalogue number N-8431, and phenylethyl ß-D-thiogalactopyranoside (PETG), catalogue number P-1692, were purchased from Invitrogen (Carlsbad, CA). The 96-well microplates, catalogue number 3635, were purchased form Corning Life Science, (Cambridge, Massachusetts). ß-galactosidase enzyme cat. # G-6008, sodium phosphate, magnesium chloride and 2-mercaptoethanol were obtained from Sigma-Aldrich Chemical Company (St Louis, Missouri). The ß-galactosidase assay was performed according to Sanbrook et.al (1). 100 ml aliquots of samples or standards diluted in distilled water were placed in each well of a 96-well microplate. The assay was initiated by the addition of 100 ml of 2X assay buffer. Assay buffer (1X) consists of 100 mM sodium phosphate, pH 7.0 1 mM MgCl2 50 mM ß-mercaptoethanol and 0.665 mg/ml ONPG in distilled water. Assay buffer was prepared previously as a 2X stock solution and stored frozen at -20°C. Lyophilized ß-galactosidase enzyme was reconstituted with distilled water to stock concentration of 500 U/ml. Enzyme dilutions were made fresh daily and stored on ice until assayed. A series of enzyme dilutions ranging from 0 to 5 U/ml of ß-galactosidase (ß-gal) were then made using distilled water as the diluent. All absorbance determinations were made at 420 nm using a PowerWave&trade HT Microplate Spectrophotometer (BioTek Instruments, Winooski, VT) with the reader controlled by Gen5 Data Analysis Software (BioTek Instruments, Winooski, VT). After the assay was initiated by the addition of the 2X assay buffer, kinetic readings were initiated immediately with absorbance determinations made every 30 seconds for a total of 60 minutes at ambient temperature.

In order to examine the effect of the inhibitor PETG on ß-gal activity a series of dilutions were made and added to the reaction mixture. Stock solutions (10 mg/ml) of PETG were made in acetonitrile and stored at -20°C until needed. Further dilutions ranging from 0 to 1000 µg/ml were then made using 1X assay buffer as the diluent. These dilutions were added to equivalent ß-gal reactions. To each well, 100 µl of 0.25 U/ml ß-gal enzyme was added along with 100 µl of the appropriate inhibitor dilution in 1X reaction buffer. As before, the assay was initiated by the addition of 100 µl of 2X reaction buffer containing ONPG as the substrate. Subsequent data reduction was performed using Gen5 software.

In order to determine Michaelis-Menten equation values, a series of dilutions of the ONPG substrate were utilized. A 20 mM stock solution of ONPG substrate in 2X reaction buffer was prepared and dilutions from 0 to 20 mM were made using 2X reaction buffer without ONPG as the diluent. Aliquots of 100 µl for each dilution were pipetted into microplate wells in replicates of 8. Reactions were initiated by the addition of 100 µl of 0.25 U/ml ß-galactosidase in water to each well and kinetic absorbance reading performed as previously described. In experiments where PETG was used in conjunction with variable ONPG substrate concentrations, reactions were performed in duplicate.

Resultate

The absorbance of enzyme concentrations ranging from 0 to 5 U/ml were read kinetically. When absorbance values are plotted as a function of time, enzyme concentrations up to 0.1 U/ml are linear for at least 60 minutes (Figure 3). Enzyme concentrations above 0.5 U/ml result in a plateau of absorbance prior to 60 minutes, with the 5 U/ml samples reaching maximal values by 2 minutes. When Vmaks values are calculated from these data and plotted against enzyme concentration using Gen5 a linear relationship is observed (Figure 4). Using a polynomial regression analysis of these data an equation describing this relationship can be used with a high degree of confidence, as the coefficient of determination (R 2 ) is 0.9998. When data from enzyme concentrations up to 1 U/ml are considered, the Vmaks response is linear. Linear regression analysis of this subset of the data results in a coefficient of determination (R 2 ) of 0.998 (data not shown).

The effect of substrate concentration on velocity was analyzed. As demonstrated in Figure 5, the velocity of the reaction increases dramatically as the ONPG substrate concentration is increased to 2 mM in the presence of 0.25 U/ml of purified ß-galactosidase enzyme. ONPG levels above 2 mM do not appreciably increase the reaction rate. An estimation of Vmaks and Km can be made from these data. Using a 4-Parameter logistic fit of the data to describe the data, Vmaks can be estimated from parameter &ldquod&rdquo (theoretical response at infinite concentration) of the equation (2). Using this method, the Vmaks was determined to be 33.4 mOD/min (Figure 5).

The Michaelis constant or Km, which represents the substrate concentration, which results in half-maximal velocity, can also be calculated using Gen5 and was determined to be 0.24 mM in this experiment.

Although the value for Km is equal to the substrate concentration ([S]) at half-maximal velocity (Vmaks/2), direct determination of Vmaks is frequently difficult to obtain experimentally. Michaelis-Menten values for Km en Vmaks have been estimated from several transformations of the original equation. These transformations include the Eadie-Hofstee and the Lineweaver-Burk plots. The Eadie-Hofstee transformation plots velocity against the velocity divided by the substrate concentration (Eq. 2)

The Lineweaver-Burk or double reciprocal transformation plots the reciprocal of velocity (1/v) against the reciprocal of substrate concentration (1/[S]) (Eq. 3). These types of plots, which generate linear regressions, allow the investigator to more easily determine Km en Vmaks. When using the Eadie-Hofstee transformation (Eq. 2) the slope is equal to &ndashKm, while Vmaks is determined from the y-intercept.

Using this transformation we determined an apparent Km of 0.1556 mM for the enzyme and a Vmaks of 34.89 mOD/min (Figure 6). Using a Lineweaver-Burk plot, Vmaks, which can be calculated from the reciprocal of the y-intercept, was determined to be 39.1 mOD/min (Figure 7). The apparent Km, which can be calculated from the slope of the line (slope = Km/Vmaks), was determined to be 0.3449 mM (Figure 7).

The effect of PETG on the ß-galactosidase enzyme activity was investigated. ß-galactosidase reactions containing equal amounts of both enzyme and ONPG substrate were read kinetically in the presence of increasing amounts of the non-hydrolyzable compound PETG. Increasing amounts of PETG result in a sigmoidal decrease in the Vmaks values for ß-galactosidase (Figure 8). Inhibitor concentrations above 1000 µg/ml result in virtually no enzyme activity, while concentrations below 0.15 µg/ml demonstrate very little inhibition.

The influence of inhibitor on the substrate concentration ([S]) reaction velocity (v) relationship was investigated. As demonstrated in Figure 8, when a constant amount of enzyme is incubated with increasing amounts of substrate in the presence of PETG, the ability to reach the maximal reaction rate (Vmaks) is diminished. When the substrate is in large excess relative to the inhibitor, Vmaks is achieved (Figure 9, red line). However, when a relatively large amount of inhibitor relative to substrate is present (Figure 9, green line) then Vmaks cannot be achieved. When Lineweaver-Burk plots are made using these data, regression lines with different slopes are observed, suggesting that the inhibitor influences the Km of the reaction (Figure 10). The linear regressions all relatively have the same y-intercept point indicating that all of the reactions have the same Vmaks. When the Vmaks values are calculated using Gen5 from the y-intercepts of the plots values of 30.94, 30.65, and 26.07 mOD/min are obtained for samples containing 0, 0.167, and 1.67 mM PETG respectively.

Bespreking

Initial experiments demonstrate the importance of reaction concentrations. Maintaining an appropriate relationship between the catalytic enzyme concentration, substrates, and incubation time is paramount in obtaining appropriate linearity in the assay. For example, the plateau of absorbance seen with the higher enzyme concentrations indicates that either shorter incubation times (e.g. 2 to 5 minutes) would provide superior linearity for these enzyme concentrations. Lower enzyme concentrations can be used to maintain linearity for 30 or 60 minute incubations. Increasing the ONPG substrate concentration is limited by the solubility of the compound in the aqueous reaction buffer and by the microplate reader absorbance limits.

The reaction kinetics for this enzyme would be expected to be linear with regard to Vmaks and enzyme concentration. Because the monomeric protein interacts with one substrate at a time, allosteric binding effects would not be observed. The tailing off of the Vmaks seen in Figure 4 at the highest enzyme concentration tested is most likely due to rapid depletion of ONPG substrate within 60 seconds (two time points). This is corroborated by the data in Figure 3, which demonstrates the rapidity of reaching maximal absorbance for high concentrations of enzyme.

When reaction velocity is plotted against substrate concentration maximal velocity Vmaks and Km can be determined. With a 4-parameter logistic fit an estimate of maximal velocity is made using parameter &ldquod&rdquo. The Michaelis constant can then be calculated by entering in the value &ldquoVmaks/2&rdquo using the interpolation function of Gen5. The resultant concentration returned is the Km. Because of the difficulty of estimating the Vmaks prior to the advent of computers, several methods were developed to determine these values using linear regression. Two such examples are the Eadie-Hofstee transformation and Lineweaver-Burk plots. Similar results were obtained for Vmaks regardless of the method. Interestingly, the average of these two means of determining Km (0.25 mM) results in a value very close to that determined from the original velocity vs. substrate concentration plot (Figure 5) which was 0.24 mM. Although the calculated Km for ONPG is higher than reported values for ß-galactosidase using lactose as the substrate (3), it is not remarkably greater.

The inhibitor PETG is a non-hydrolyzable substrate for ß-gal. The substitution of a thiol linkage for a hydroxyl group eliminates hydrolysis (Figure 3). Experiments with PETG suggest that the inhibitor has an equal affinity for the enzyme as the substrate ONPG. The molar concentration at which PETG begins to affect enzyme activity is very close to the concentration of the ONPG substrate indicating similar affinities. If the enzyme had a greater affinity for ONPG than PETG then inhibition would have required proportionally more of the inhibitor to elicit the same response.

These data presented also indicate that PETG is a competitive rather than a non-competitive inhibitor of ß-galactosidase. Measurements of the reaction rates at different concentrations of substrate and inhibitor serve to distinguish between competitive and non-competitive inhibition. Competitive inhibitors compete for enzyme binding sites and as such, a large excess of substrate to inhibitor will result in reaction rates similar to samples that do not contain inhibitor. This would result in Vmaks values that are equivalent to that of enzyme without inhibitors present (Figure 10). The discrepancy between the calculated Vmaks for the 1.67 mM PETG samples and controls (0 mM PETG) is the result of ONPG not being in sufficient excess. Noncompetitive inhibitors would show different Vmaks values regardless of the substrate concentration (3). PETG also presents chemical structure very similar to ONPG, suggesting that it will bind to the active binding site of the ß-galactosidase enzyme. In the past such kinetic determinations have been performed using a conventional spectrophotometer, which usually entails the use of matched cuvettes to perform the analysis, resulting in a very low throughput. The ability to use the PowerWave&trade HT Microplate Reader to perform this analysis allows this routine procedure to be performed on 96 samples in a matter of seconds leading to a tremendous increase in productivity and throughput. These data presented in this report also demonstrate the utility of Gen5 Data Analysis Software to perform routine enzyme kinetic studies. Automatic determination of values such as Vmaks and Km allows the end user flexibility in regards to data reduction of kinetic assays.

Verwysings

(1) Sanbrook J. E.F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY.

(2) BioTek Instruments, Application Note &ldquoBioTek&rsquos 4-Parameter Logistic Fit&rdquo, Winooski, Vermont.


Inleiding

Cell-to-cell heterogeneity is a feature of processes of great interest in basic and disease research (for example, cancer metastasis 1, 2 and drug responses of tumor cells 3, 4, 5 ). Understanding of these processes can benefit greatly from single-cell measurements, especially those that can relate differences in function or phenotype and the extracellular microenvironment to variations in intracellular state of individual cells. 6, 7 Traditional cellular assays, however, due to sensitivity limitations, measure average properties of large numbers (usually 10 3 � 6 ) of cells. Such measurements mask the differences between individual cells. 3, 8, 9

Several current techniques allow assaying cells individually for differing types of information. Harnessing nucleic acid amplification and sequencing technologies, a number of assays measure genetic information and gene expression from single cells. 10, 11, 12, 13, 14 Microfluidic realizations of these assays achieve high sensitivity and throughput. Most such techniques, however, require cells in suspension. Placing adherent cells in suspension destroys information about tissue context, and makes it difficult to relate measured variations to this context or to phenotypic differences observable only when cells are adherent to a substrate.

Since even genetically identical cells may respond differently to the same cues, 3, 4, 5 as many additional layers of regulation determine cellular behavior, single-cell measurement at the protein level is desirable. Assaying for protein levels, localization or activity from single cells faces additional challenges over gene-based assays, not only due to the lack of a generic amplification scheme but also because proteins are dynamic on a shorter time-scale (and thus more quickly responsive to unwanted perturbations introduced by the assay method). The ability to make measurements of signaling proteins, for example kinases, at the single-cell level is especially relevant to the major goal of understanding how a cell processes information from external cues to generate a response. This can help in understanding how to alter cell outcomes in a controlled manner, which has great implications for therapeutics. 3, 4, 5 Thus a means to obtain a clear picture of signaling events in a cell, and ideally clarify the connection between signaling and phenotype for a particular cell while knowing its external context, is desirable.

For examining cell signaling events, protein activity is more relevant than protein level, reporting more directly on actions occurring in the cell. However, levels of proteins and protein post-translational modification (PTM) states, which are less challenging to measure, are often used as proxies for the actual activity. Flow and phospho-flow cytometry, as well as mass cytometry, 15 enable high-throughput multiplexed measurements of these from single cells, but have several drawbacks. A first problem is the assumption that the phosphorylation state of a kinase is a good proxy for its activity. Because of the complex and incompletely understood nature of signaling regulation via phosphorylation (and other protein PTMs), this is not necessarily the case, including for heavily-studied kinases such as Akt. 16, 17, 18 A corollary issue is that even for cases in which an identified PTM state definitively governs activity level, this can often involve multiple PTMs which are typically not quantified concomitantly. 19 A second challenge is that these assays generally rely on the existence of high-quality antibodies specific for the protein or protein PTM of interest. Third, such assays do not easily permit association of the molecular signaling measurements with a phenotypic characterization of any given cell (other than via surrogate molecular markers), so a goal of ascertaining relationships between signaling state and phenotypic behavior on a cell-to-cell level is elusive. Finally, these assays, as well as microfluidic approaches to single-cell protein-level measurement, 20 predominantly require a suspension step before measurement can be undertaken, which is likely to affect signaling.

Direct measurements from single adherent cells, at the genetic or protein level, without perturbing the cells and their extracellular milieu are therefore highly desirable, but limited means currently exist to accomplish this. 21 For the particularly challenging case of measuring protein activities, only a few methods exist, each with its difficulties. Injected sensors present challenges with specificity and stability, and injection disrupts the cell. 22 Live-cell imaging using genetically encoded reporters can provide dynamic information on protein levels and localization or on activity directly, 23 but such reporters are difficult to multiplex, and genetic manipulation presents the risk of affecting the system under study. The approach is also highly resource-intensive, and presents challenges in obtaining quantitative measurements. 24

We have previously used a microfluidic concentrator device to fluorescently monitor specific kinase activities from unfractionated cell lysates using peptide sensors incorporating the non-natural amino acid Sox, a chelation-enhanced fluorophore. 25, 26 This achieved the sensitivity to measure kinase activity from an amount of bulk-level cell lysate estimated to be equivalent to a few cells' worth. To perform this assay directly on a single cell selected from an adherent cell population, however, requires development of the novel capability to selectively lyse an individual adherent cell, and capture its contents while limiting dilution to retain assay sensitivity. Such a capability would open up the sensitivity, throughput and automation advantages conferred by microfluidics to a host of biochemical assays and enable their use at the single-cell level while retaining the ability to integrate these measurements with existing data from standard assay platforms.

In the current work, we have developed an integrated microfluidic probe device that assays the contents of single adherent cells from standard tissue culture. It achieves selective single cell lysis by hydrodynamic confinement of a lysis buffer at its tip and captures the contents of single cells and mixes them with assay reagents in nanoliter-scale integrated chambers to perform high sensitivity single-cell assays. We demonstrate the selective lysis and capture of the contents of single adherent cells without disrupting neighboring cells even in near-confluent cultures. We then use the device to measure kinase and housekeeping protein activities, simultaneously or separately, directly from single hepatocellular carcinoma (HepG2) cells in adherent tissue culture. We demonstrate that this approach can characterize biological heterogeneity in Akt kinase activity levels among cells under insulin stimulation.


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