Ukuhlaziya ama-leukocyte egazini lonke kuwukuhlola okuvamile elebhu yasemtholampilo noma ebhange legazi.Ukugxila nokusebenza kwama-leukocyte kuyinkomba ebalulekile njengokulawulwa kwekhwalithi yokugcinwa kwegazi.Ngaphandle kwe-leukocyte, igazi eliphelele liqukethe inani elikhulu lama-platelet, amangqamuzana egazi abomvu, noma imfucumfucu yamangqamuzana, okwenza kube nzima ukuhlaziya igazi eliphelele ngokuqondile ngaphansi kwe-microscope noma ikhawunta ye-cell field ekhanyayo.Izindlela ezijwayelekile zokubala amangqamuzana amhlophe egazi zibandakanya inqubo ye-RBC lysis, edla isikhathi.
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® Rigel.
I-Countstar Rigel iyisixazululo esifanelekile sokuhlolwa kwezimpawu zamangqamuzana amaningi ayinkimbinkimbi, okwenza sikwazi ukuhlaziya ngokushesha amangqamuzana egazi amhlophe egazini lonke.
Inqubo Yokuhlola:
1.Thatha u-20 µl wesampula yegazi bese uhlanza isampula ku-180 µl we-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 Ufaka Isampula ID yale sampuli.
7.Khetha i-Dilution ratio, Uhlobo Lweseli, ukuchofoza u-'Run' ukuze uqale ukuhlolwa.
Isexwayiso: I-AO ne-PI iwumdlavuza ongaba khona.Kunconywa ukuthi opharetha agqoke izinto zokuzivikela (i-PPE) ukuze agweme ukuthintana ngqo nesikhumba namehlo.
Umphumela:
1. Isithombe seNsimu Ekhanyayo yegazi eliphelele
Emfanekisweni ogqamile wegazi lonke, ama-WBC awabonakali phakathi kwamangqamuzana egazi abomvu.(Umfanekiso 1)
Umfanekiso 1 Isithombe senkundla ekhanyayo segazi eliphelele.
2. Isithombe se-Fluorescence segazi eliphelele
Udayi we-AO kanye ne-PI womabili amabala e-DNA ku-nucleus yeseli yamaseli.Ngakho-ke, ama-Platelets, amangqamuzana egazi abomvu, noma ama-debris amaselula awakwazi ukuphazamisa ukuhlushwa kwe-leukocyte kanye nomphumela wokusebenza.Ama-leukocyte aphilayo (Aluhlaza) nama-leukocyte afile (Abomvu) abonakala kalula ezithombeni ze-fluorescence.(Umfanekiso 2)
Umfanekiso 2 Izithombe ze-Fluorescence zegazi eliphelele.(A).Isithombe se-AO Channel;(B) Isithombe se-PI Channel;(C) Hlanganisa izithombe ze-AO ne-PI Channel.
3. Ukugxila nokusebenza kwama-leukocyte
Isofthiwe ye-Countstar FL ibala ngokuzenzakalelayo amaseli ezigaba ezintathu zegumbi futhi ibale inani eliyisilinganiso sengqikithi yokubala kwamaseli e-WBC (1202), ukugxila (1.83 x 106 amaseli/ml), kanye nokusebenza okungu-% (82.04%).Zonke izithombe zegazi nedatha kungathunyelwa kalula njenge-PDF, Isithombe noma i-Excel ukuze kuhlaziywe okwengeziwe noma kugcinwe idatha.
Umfanekiso 3 Isithombe-skrini se-Countstar Rigel Software
