Inkqubo ye-Countstar idibanisa i-cytometer yomfanekiso kunye ne-counter counter kwisixhobo esinye sebhentshi.Esi sicelo siqhutywa, i-compact, kunye ne-automated cell imaging system ibonelela ngesisombululo se-in-one sophando lweseli yomhlaza, kubandakanywa ukubala kweeseli, ukusebenza (AO / PI, trypan blue), apoptosis (Annexin V-FITC / PI), iseli umjikelo (PI), kunye nokudluliselwa kwe-GFP/RFP.
Abstract
Umhlaza ngoyena nobangela wokufa kwihlabathi liphela, kwaye uphuhliso lweendlela ezintsha zonyango lomhlaza lubaluleke kakhulu.Iseli yomhlaza yinto esisiseko yophando yomhlaza, ulwazi olwahlukeneyo kufuneka luvavanywe kwiseli yomhlaza.Lo mmandla wophando ufuna uhlalutyo lweeseli olukhawulezayo, oluthembekileyo, olulula noluneenkcukacha.Inkqubo ye-Countstar ibonelela ngeqonga lesisombululo esilula sohlalutyo lweseli yomhlaza.
Study Cancer Cell Apoptosis ngu Countstar Rigel
Uvavanyo lwe-apoptosis lusetyenziswa rhoqo kwiilabhoratri ezininzi ngeenjongo ezahlukeneyo ukusuka kuvavanyo lwempilo yeenkcubeko zeeseli ukuya kuphononongo lwetyhefu lwephaneli yeekhompawundi.
I-Apoptosis assay luhlobo olusetyenziselwa ukumisela ipesenti ye-apoptosis yeeseli nge-Annexin V-FITC / PI staining method.I-Annexin V ibophelela kwi-phosphatidylserine (PS) kunye neseli ye-apoptosis yokuqala okanye iseli ye-necrosis.I-PI ingena kuphela kwiiseli ze-apoptotic ze-necrotic/inqanaba kade kakhulu.(Umfanekiso 1)
A: Early apoptosis Annexin V (+), PI (-)
B:Late apoptosis Annexin V (+), PI (+)
Umzobo1: Iinkcukacha ezandisiweyo ze-Countstar Rigel imifanekiso (5 x magnification) yeeseli ze-293, ziphathwe nge-Annexin V FITC kunye ne-PI
Cell Cycle Analysis of Cancer Cell
Umjikelo weseli okanye umjikelo wokwahlulwahlulwa kweeseli luluhlu lweziganeko ezenzeka kwiseli ekhokelela ekwahlulweni kwayo kunye nokuphindaphinda i-DNA yayo (i-DNA replication) ukuvelisa iiseli ezimbini zeentombi.Kwiiseli ezinenucleus, njengakwi-eukaryotes, umjikelo weseli ukwahlulwe ngokwamaxesha amathathu: i-interphase, i-mitotic (M) phase, kunye ne-cytokinesis.IPropidium iodide (PI) yidayi engcolisa inyukliya esetyenziswa rhoqo ukulinganisa umjikelo weseli.Ngenxa yokuba idayi ayikwazi ukungena kwiiseli eziphilayo, iiseli zilungiswa nge-ethanol ngaphambi kokungcoliswa.Zonke iiseli emva koko zinebala.Iiseli ezilungiselela ulwahlulo ziya kuqulatha izixa ezikhulayo ze-DNA kwaye zibonise ngokulinganayo ukwanda kwe-fluorescence.Umahluko kwi-fluorescence intensity isetyenziselwa ukumisela ipesenti yeeseli kwisigaba ngasinye somjikelezo weseli.I-Countstar inokuwuthatha umfanekiso kwaye iziphumo ziya kuboniswa kwi-software ye-FCS express.(Umfanekiso 2)
Umzobo 2: I-MCF-7 (A) kunye ne-293T (B) yahlanjululwa nge-cell cycle Detection Kit nge-PI, iziphumo zichongwe ngu-Countstar Rigel, kwaye zihlalutywe yi-FCS express.
Ukusebenza kunye neGFP Transfection Detemination in Cell
Ngexesha le-bioprocess, i-GFP isoloko isetyenziselwa ukuxubha kunye neprotheni edibeneyo njengesalathisi.Ukumisela i-fluorescent ye-GFP ingabonakalisa inkcazo yeprotheyini ekujoliswe kuyo.I-Countstar Rigel ibonelela ngovavanyo olukhawulezayo nolulula lovavanyo losulelo lwe-GFP kunye nokusebenza.Iiseli zadyojwa ngePropidium iodide (PI) kunye neHoechst 33342 ukuchaza inani leeseli ezifileyo kunye neeseli ezipheleleyo.I-Countstar Rigel ibonelela ngendlela ekhawulezayo, yobungakanani bokuvavanya ukusebenza kakuhle kokubonakaliswa kwe-GFP kunye nokusebenza ngexesha elinye.(Umfanekiso 4)
Umzobo 4: Iiseli zibekwe kusetyenziswa i-Hoechst 33342 (ebhlowu) kwaye ipesenti ye-GFP ebonakalisa iiseli (eluhlaza) inokumiselwa ngokulula.Iseli engasebenziyo ingcoliswe yipropidium iodide (PI; ebomvu).
Ukusebenza kunye nokubala kweeSeli
I-AO/PI Ukubalwa kwe-Dual-fluoresces luhlobo lovavanyo olusetyenziselwa ukufumanisa ukugxininiswa kweeseli, ukusebenza.Yahlulahlulwe yaba lilayini yokubala kunye nokubalwa kweeseli eziphambili ngokohlobo lweeseli olwahlukileyo.Isisombululo siqulethe i-green-fluorescent nucleic acid stain, i-acridine orange, kunye ne-redfluorescent nucleic acid stain, i-propidium iodide.I-Propidium iodide yidayi yokukhutshwa kwenwebu engena kuphela kwiiseli ezineembrane eziphazamisekileyo ngelixa i-acridine orange ingena 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 ezinemembrane ezingaguqukiyo zingcolisa i-fluorescent eluhlaza kwaye zibalwa njengeziphilayo, ngelixa iiseli ze-nucleated ezineminwebu eyonakeleyo zibala umbala obomvu we-fluorescent kwaye zibalwa njengezifileyo xa kusetyenziswa inkqubo ye-Countstar Rigel.Izinto ezingenayo i-nucleated ezifana neeseli ezibomvu zegazi, iiplatelets kunye ne-debris azikhuli kwaye azihoywa yi-software ye-Countstar Rigel.(Umfanekiso 5)
Umzobo 5: I-Countstar iye yalungisa indlela yokungcolisa i-dual-fluorescence ukuze kube lula, ukuzimisela okuchanekileyo koxinzelelo lwe-PBMC kunye nokusebenza.Iisampulu ezingcoliswe nge-AO/PI zinokuhlalutywa kunye ne-Counstar Rigel
