Amaseli egazi e-peripheral mononuclear (PBMCs) avame ukucutshungulwa ukuze ahlukaniswe negazi eliphelele nge-density gradient centrifugation.Lawo maseli aqukethe ama-lymphocyte (ama-T cell, ama-B cell, ama-NK cell) nama-monocyte, avame ukusetshenziswa emkhakheni we-immunology, ukwelashwa kwamaseli, izifo ezithathelwanayo nokuthuthukiswa komgomo.Ukuqapha nokuhlaziya ukusebenza kahle nokugxila kwe-PBMC kubalulekile kumalabhorethri omtholampilo, ucwaningo oluyisisekelo lwesayensi yezokwelapha kanye nokukhiqizwa kwamaseli omzimba.
Umdwebo 1. I-PBMC Eyodwa egazini elisha eline-Density gradient centrifugation
Ukubala kwe-AOPI Dual-fluoresces wuhlobo lokuhlola olusetshenziselwa ukuthola ukugcwala kwamaseli nokusebenza.Isixazululo inhlanganisela ye-acridine orange (ibala eliluhlaza-fluorescent nucleic acid) ne-propidium iodide (ibala elibomvu le-fluorescent nucleic acid).I-Propidium iodide (PI) wudayi wokukhishwa kwe-membrane ongena kuphela kumaseli anolwelwesi olusengozini, kuyilapho i-acridine orange ikwazi ukungena kuwo wonke amangqamuzana kubantu.Uma bobabili odayi bekhona ku-nucleus, i-propidium iodide ibangela ukuncipha kwe-acridine orange fluorescence ngokudluliswa kwe-fluorescence resonance energy (FRET).Ngenxa yalokho, amaseli angama-nucleated anolwelwesi olungaguquki angcolisa okuluhlaza okotshani futhi abalwa njengaphilayo, kuyilapho amaseli e-nucleated anolwelwesi olusengozini angcolisa kuphela okubomvu kwe-fluorescent futhi abalwa njengafile lapho kusetshenziswa uhlelo lwe-Countstar® FL.Izinto ezingezona i-nucleated njengamaseli egazi abomvu, ama-platelet kanye nemfucumfucu akushintshi futhi azinakwa isofthiwe ye-Countstar® FL.
Inqubo Yokuhlola:
1.Nciphisa isampula ye-PBMC ekugxiliseni oku-5 okuhlukene nge-PBS;
2.Engeza isixazululo esingu-12µl AO/PI kusampula engu-12µl, exutshwe kahle nepipette;
3.Dweba ingxube engu-20µl kusilayidi segumbi;
4.Vumela amaseli ukuthi ahlale ekamelweni cishe iminithi elingu-1;
5.Faka i-slide ensimbini ye-Countstar FL;
6.Khetha ukulinganisa kwe-“AO/PI Viability”, bese uvivinya nge-Countstar FL.
Isexwayiso: I-AO ne-PI iwumdlavuza ongaba khona.Kunconywa ukuthi opharetha agqoke izinto zokuzivikela (i-PPE) ukuze agweme ukuthintana ngqo nesikhumba namehlo.
Umphumela:
1.Izithombe ze-Bright Field kanye ne-Fluorescence ye-PBMC
Udayi we-AO kanye ne-PI womabili amabala e-DNA ku-nucleus yeseli yamaseli.Ngakho-ke, ama-Platelets, amangqamuzana egazi abomvu, noma udoti wamaselula awakwazi ukuthikameza ukugxiliswa kwe-PBMC nomphumela wokusebenza.Amaseli aphilayo, amaseli afile kanye nemfucumfucu kungahlukaniswa kalula ngesisekelo sezithombe ezikhiqizwe i-Countstar FL (Umfanekiso 1).
Umfanekiso 2.Izithombe ze-Bright Field kanye ne-Fluorescence ye-PBMC
2.Ukugxila kanye nokusebenza kwe-PBMC
Amasampuli e-PBMC ahlanjululwe izikhathi ezingu-2, 4, 8 kanye ne-16 nge-PBS, kwase kuthi lawo masampuli afakwa ngengxube kadayi ye-AO/PI futhi ahlaziywa yi-Countstar FL ngokulandelanayo.Umphumela wokugxilisa ingqondo nokusebenza kwe-PBMC uboniswa njengesibalo esingezansi:
