Iiseli ze-peripheral blood mononuclear (PBMCs) zihlala zisetyenzwa ukuze zahlukane negazi elipheleleyo nge- density gradient centrifugation.Ezo seli ziqulathe lymphocytes (iiseli T, iiseli B, NK iiseli) kunye monocytes, ngokuqhelekileyo esetyenziswa entsimini immunology, unyango cell, isifo esosulelayo kunye nophuhliso lokugonya.Ukubeka iliso kunye nokuhlalutya ukusebenza kunye noxinzelelo lwe-PBMC lubalulekile kwiilabhoratri zeklinikhi, uphando olusisiseko lwesayensi yezonyango kunye nokuveliswa kweeseli zomzimba.
Umzobo 1. I-PBMC ekwanti ukusuka kwigazi elitsha eline-Density gradient centrifugation
I-AOPI Ubalo lwe-Dual-fluoresces luhlobo lovavanyo olusetyenziselwa ukufumanisa ukugxila kweeseli kunye nokusebenza.Isisombululo siyindibaniselwano ye-acridine orange (ibala eliluhlaza-fluorescent nucleic acid) kunye nepropidium iodide (ibala elibomvu-elikhanyayo le-nucleic acid).I-Propidium iodide (PI) yidayi yokukhutshwa kwe-membrane engena kuphela kwiiseli ezineembrane eziphazamisekileyo, ngelixa i-acridine orange iyakwazi ukungena kuzo zonke iiseli kubemi.Xa zombini iidayi zikhona kwi-nucleus, i-propidium iodide ibangela ukunciphisa i-acridine orange fluorescence nge-fluorescence resonance energy transfer (FRET).Ngenxa yoko, iiseli ze-nucleated ezine-membrane ezingaguqukiyo zingcolisa i-fluorescent eluhlaza kwaye zibalwa njengeziphilayo, kanti iiseli ze-nucleated ezinemibhobho ephazamisekileyo kuphela zibala umbala obomvu we-fluorescent kwaye zibalwa njengezifileyo xa usebenzisa inkqubo ye-Countstar® FL.Izinto ezingenayo i-nucleated ezifana neeseli ezibomvu zegazi, iiplatelet kunye ne-debris azikhuli kwaye azihoywa yi-software ye-Countstar® FL.
Inkqubo yoMfuniselo:
I-1.Dilute isampuli ye-PBMC kwii-5 ezahlukeneyo zogxininiso kunye ne-PBS;
2.Yongeza isisombululo se-12µl AO/PI kwisampulu ye-12µl, exutywe ngobunono kunye nepipette;
3.Zoba 20µl umxube kwisilayidi segumbi;
4.Vumela iiseli ukuba zihlale kwigumbi malunga nomzuzu omnye;
5.Tyala isilayidi kwisixhobo se-Countstar FL;
6.Khetha i-assay "AO / PI Viability", emva koko uvavanye ngu-Countstar FL.
Isilumkiso: I-AO kunye ne-PI inokuba yi-carcinogen.Kuyacetyiswa ukuba umqhubi anxibe izixhobo zokuzikhusela (PPE) ukuthintela ukunxibelelana ngokuthe ngqo nolusu kunye namehlo.
Isiphumo:
1.Imifanekiso ye-Bright Field kunye neFluorescence ye-PBMC
Idayi ye-AO kunye ne-PI zombini zibala i-DNA kwinucleus yeeseli.Ke ngoko, iiPlatelets, iiseli ezibomvu zegazi, okanye i-debris yeselula ayikwazi ukuchaphazela uxinzelelo lwe-PBMC kunye nesiphumo sokusebenza.Iiseli eziphilayo, iiseli ezifileyo kunye ne-debris ziyakwazi ukwahlula ngokulula isiseko kwimifanekiso eyenziwe yi-Countstar FL (Umfanekiso 1).
Umzobo 2.Umfanekiso oqaqambileyo webala kunye neFluorescence ye-PBMC
2.Ukugxila kunye nokusebenza kwe-PBMC
Iisampuli ze-PBMC zahlanjululwa kwi-2, i-4, i-8 kunye ne-16 ngamaxesha kunye ne-PBS, emva koko ezo sampuli zixutywe ngomxube wedayi we-AO / PI kwaye zihlalutywe yi-Countstar FL ngokulandelanayo.Isiphumo sokugxila kunye nokusebenza kwe-PBMC kuboniswa njengamzobo ungezantsi:
