Uhlelo lwe-Countstar luhlanganisa i-cytometer yesithombe nekhawunta yeseli ibe ithuluzi elilodwa elingaphezulu kwebhentshi.Lolu hlelo lokusebenza oluqhutshwa uhlelo lokusebenza, oluhlangene, futhi oluzenzakalelayo lokucabanga kwamaseli luhlinzeka ngesixazululo esihlangene socwaningo lwamaseli omdlavuza, okuhlanganisa ukubala kwamaseli, ukusebenza kahle (AO/PI, trypan blue), apoptosis (Annexin V-FITC/PI), iseli. umjikelezo (PI), kanye nokudluliselwa kwe-GFP/RFP.
Abstract
Umdlavuza ungenye yezimbangela eziyinhloko zokufa emhlabeni wonke, futhi ukuthuthukiswa kwezindlela ezintsha zokwelapha umdlavuza kubaluleke kakhulu.I-Cancer cell iwucwaningo oluyisisekelo lwento yomdlavuza, imininingwane ehlukahlukene idinga ukucutshungulwa engqamuzaneni lomdlavuza.Le ndawo yocwaningo idinga ukuhlaziywa kwamaseli okusheshayo, okuthembekile, okulula nokunemininingwane.Uhlelo lwe-Countstar lunikeza inkundla yesixazululo esilula sokuhlaziya amangqamuzana omdlavuza.
Funda I-Cancer Cell Apoptosis ngu-Countstar Rigel
Izivivinyo ze-apoptosis zisetshenziswa njalo kumalabhorethri amaningi ngezinjongo ezihlukahlukene kusukela ekuhloleni impilo yamasiko amaseli kuya ekuhloleni ubuthi bephaneli lezinhlanganisela.
I-Apoptosis assay iwuhlobo olusetshenziselwa ukunquma iphesenti le-apoptosis lamaseli nge-Annexin V-FITC/PI staining method.I-Annexin V ibophezela ku-phosphatidylserine (PS) ngeseli ye-apoptosis yokuqala noma iseli ye-necrosis.I-PI ingena kuphela kumaseli e-apoptotic e-necrotic/asekupheleni kakhulu.(Umfanekiso 1)
A: I-apoptosis yangaphambi kwesikhathi i-Annexin V (+), PI (-)
B:Late apoptosis Annexin V (+), PI (+)
Umfanekiso1: Imininingwane enwetshiwe yezithombe ze-Countstar Rigel (5 x ukukhuliswa) zamaseli angama-293, aphathwe nge-Annexin V FITC ne-PI.
I-Cell Cycle Analysis of Cancer Cell
Umjikelezo weseli noma umjikelezo wokuhlukaniswa kwamaseli uchungechunge lwezehlakalo ezenzeka kuseli okuholela ekuhlukaneni nasekuphindaphindeni kwe-DNA yayo (i-DNA replication) ukuze kukhiqizwe amaseli amantombazane amabili.Emangqamuzaneni ane-nucleus, njengaku-eukaryote, umjikelezo wamaseli nawo uhlukaniswe izinkathi ezintathu: i-interphase, isigaba se-mitotic (M), ne-cytokinesis.I-Propidium iodide (PI) udayi onamabala we-nuclear ovame ukusetshenziswa ukukala umjikelezo wamaseli.Ngenxa yokuthi udayi awukwazi ukungena kumaseli aphilayo, amaseli ahlanganiswa ne-ethanol ngaphambi kokungcoliswa.Wonke amaseli abe eseba namabala.Amaseli alungiselela ukwehlukana azoqukatha amanani akhulayo e-DNA futhi abonise i-fluorescence ekhuphukile ngokulinganayo.Umehluko kumfutho we-fluorescence usetshenziselwa ukunquma iphesenti lamaseli esigabeni ngasinye somjikelezo weseli.I-Countstar ingathwebula isithombe futhi imiphumela izoboniswa ku-software ye-FCS express.(Umfanekiso 2)
Umfanekiso 2: I-MCF-7 (A) kanye ne-293T (B) yayingcoliswe i-cell cycle Detection Kit ene-PI, imiphumela yanqunywa ngu-Countstar Rigel, futhi yahlaziywa yi-FCS express.
Ukusebenza kanye Nokunqunywa Kokudluliselwa Kwe-GFP Kuseli
Ngesikhathi se-bioprocess, i-GFP ivame ukusetshenziselwa ukuhlanganisa amaprotheni ahlangene njengenkomba.Thola ukuthi i-GFP fluorescent ingabonisa isisho sephrotheni esiqondiwe.I-Countstar Rigel inikezela ngesivivinyo esisheshayo nesilula sokuhlola ukudluliselwa kwe-GFP kanye nokusebenza.Amaseli agcotshwe nge-Propidium iodide (PI) kanye ne-Hoechst 33342 ukuze achaze inani lamaseli afile kanye nengqikithi yamaseli.I-Countstar Rigel inikezela ngendlela esheshayo, yobuningi yokuhlola ukusebenza kahle kokuvezwa kwe-GFP nokusebenza ngesikhathi esifanayo.(Umfanekiso 4)
Umfanekiso 4: Amaseli atholakala kusetshenziswa i-Hoechst 33342 (eluhlaza okwesibhakabhaka) futhi iphesenti lamaseli aveza i-GFP (aluhlaza) linganqunywa kalula.Amaseli angenakukwazi ukuphila angcoliswe i-propidium iodide (PI; ebomvu).
Ukusebenza nokubala kwamaseli
Ukubala kwe-AO/PI Dual-fluoresces wuhlobo lokuhlola olusetshenziselwa ukuthola ukugcwala kwamaseli, ukusebenza kahle.Ihlukaniswe ngokubala komugqa wamaseli nokubala kwamaseli okuyisisekelo ngokohlobo lwamaseli oluhlukene.Isixazululo siqukethe inhlanganisela ye-green-fluorescent nucleic acid stain, i-acridine orange, kanye ne-redfluorescent nucleic acid stain, i-propidium iodide.I-Propidium iodide ingudayi wokukhishwa ulwelwesi olungena kuphela kumaseli anolwelwesi olusengozini kuyilapho i-acridine orange ingena kuwo wonke amaseli esibalweni.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.Izinto ezingezona i-nucleated njengamaseli abomvu egazi, ama-platelet kanye nemfucumfucu akushintshi futhi kuzitshwa isofthiwe ye-Countstar Rigel.(Umfanekiso 5)
Umfanekiso 5: I-Countstar ithuthukise indlela yokwenza amabala e-dual-fluorescence ukuze kube lula, ukuzimisela okunembile kokugxilisa ingqondo nokusebenza kwe-PBMC.Amasampula angcoliswe nge-AO/PI angahlaziywa nge-Counstar Rigel
