Lisele tsa peripheral blood mononuclear cell (PBMCs) hangata li sebetsoa hore li arohane le mali a felletseng ka density gradient centrifugation.Lisele tseo li na le li-lymphocyte (lisele tsa T, lisele tsa B, lisele tsa NK) le li-monocyte, tse atisang ho sebelisoa tšimong ea immunology, phekolo ea lisele, mafu a tšoaetsanoang le nts'etsopele ea ente.Ho beha leihlo le ho sekaseka ho sebetsa le ho tsepamisa maikutlo ha PBMC ho bohlokoa bakeng sa lilaboratori tsa bongaka, lipatlisiso tsa mantlha tsa mahlale a bongaka le tlhahiso ea lisele tsa 'mele.
Setšoantšo sa 1. PBMC e Isolated ho tsoa maling a foreshe a nang le Density gradient centrifugation
Ho bala ha AOPI Dual-fluoresces ke mofuta oa liteko o sebelisetsoang ho bona hore na lisele li sebetsa joang.Tharollo ke motsoako oa acridine orange (letheba la nucleic acid e tala-fluorescent) le propidium iodide (letheba le khubelu-fluorescent nucleic acid).Propidium iodide (PI) ke dae e sa kenyelletsoeng lera e kenang feela liseleng tse nang le li-membrane tse senyehileng, athe acridine orange e khona ho kenella liseleng tsohle ho baahi.Ha lidae ka bobeli li le teng khubung, propidium iodide e baka phokotso ea acridine orange fluorescence ka fluorescence resonance energy transfer (FRET).Ka lebaka leo, lisele tse nang le li-nucleated tse nang le li-membrane tse sa tsitsang li senya botala ba fluorescent 'me li baloa li phela, athe lisele tsa nucleated tse nang le li-membrane tse senyehileng li senya bofubelu ba fluorescent feela' me li baloa li shoele ha li sebelisa tsamaiso ea Countstar® FL.Lintho tse se nang nucleated tse kang lisele tse khubelu tsa mali, li-platelet le litšila ha li na fluoresce 'me li hlokomolohuoa ke software ea Countstar® FL.
Mokhoa oa Teko:
1. Hlakola sampole ea PBMC ka har'a likhakanyo tse 5 tse fapaneng le PBS;
2.Eketsa tharollo ea 12µl AO/PI ho sampole ea 12µl, e kopantsoeng le pipette ka bonolo;
3.Thala motsoako oa 20µl ka kamoreng ea slide;
4.Lumella lisele hore li lule ka kamoreng ho pota 1 motsotso;
5.Senya selaete ho sesebelisoa sa Countstar FL;
6.Khetha tlhahlobo ea "AO/PI Viability", ebe u lekoa ke Countstar FL.
Tlhokomeliso: AO le PI ke ntho e ka bakang kankere.Ho khothaletsoa hore motho ea sebetsang a apare lisebelisoa tsa ho itšireletsa (PPE) ho qoba ho kopana ka kotloloho le letlalo le mahlo.
Sephetho:
1.Bright Field le litšoantšo tsa Fluorescence tsa PBMC
Dae ea AO le PI ka bobeli ke matheba a DNA khubung ea lisele.Ka hona, li-Platelets, lisele tse khubelu tsa mali, kapa litšila tsa cellular ha li khone ho ama mahloriso a PBMC le sephetho sa ts'ebetso.Lisele tse phelang, lisele tse shoeleng le litšila li ka khetholloa habonolo motheong oa litšoantšo tse hlahisoang ke Countstar FL (Setšoantšo sa 1).
Setšoantšo sa 2.Litšoantšo tsa Tšimo e Khanyang le Fluorescence tsa PBMC
2.Maikutlo le Ts'ebetso ea PBMC
Mehlala ea PBMC e ile ea hlapolloa ka makhetlo a 2, 4, 8 le 16 ka PBS, eaba lisampole tseo li kenngoa ka motsoako oa dae oa AO/PI 'me tsa hlahlojoa ke Countstar FL ka ho latellana.Sephetho sa ho tsepamisa maikutlo le ho sebetsa ha PBMC se bonts'oa joalo ka setšoantšo se ka tlase:
