Hoʻohui ka ʻōnaehana Countstar i ka cytometer kiʻi a me ka helu kelepona i loko o kahi mea kani-luna hoʻokahi.Hāʻawi kēia ʻōnaehana kiʻi kiʻi i hoʻohana ʻia, paʻa, a me ka automated cell imaging solution no ka noiʻi cell cancer, me ka helu cell, viability (AO/PI, trypan blue), apoptosis (Annexin V-FITC/PI), cell. pōʻai (PI), a me ka hoʻololi GFP/RFP.
ʻĀpana
ʻO ka maʻi kanesa kekahi o nā kumu nui o ka make ma ka honua holoʻokoʻa, a he mea nui ka hoʻomohala ʻana i nā ʻano hana lapaʻau hou.ʻO ka cell cancer ka mea noiʻi kumu o ka maʻi kanesa, pono e loiloi ʻia nā ʻike like ʻole mai ka cell cancer.Pono kēia wahi noiʻi i ka wikiwiki, hilinaʻi, maʻalahi a me nā kikoʻī kikoʻī.Hāʻawi ka ʻōnaehana Countstar i kahi kahua hoʻonā maʻalahi no ka nānā ʻana i nā cell cancer.
E aʻo i ka Cancer Cell Apoptosis na Countstar Rigel
Hoʻohana mau ʻia ka hoʻāʻo ʻana o Apoptosis i loko o nā hale hana he nui no nā kumu like ʻole mai ka loiloi ʻana i ke olakino o nā moʻomeheu cell a hiki i ka loiloi ʻana i ka ʻawaʻawa o kahi hui pūhui.
ʻO ka ho'āʻo apoptosis kahi ʻano i hoʻohana ʻia no ka hoʻoholo ʻana i ka pākēneka apoptosis o nā cell e ka Annexin V-FITC/PI staining method.Hoʻopili ʻo Annexin V i ka phosphatidylserine (PS) me ka cell apoptosis mua a i ʻole necrosis cell.Komo wale ʻo PI i nā cell apoptotic necrotic/hope loa.(Kiʻi 1)
A: Early apoptosis Annexin V (+), PI (-)
B: Late apoptosis Annexin V (+), PI (+)
Kiʻi 1: Nā kikoʻī i hoʻonui ʻia o nā kiʻi Countstar Rigel (5 x hoʻonui) o 293 cell, mālama ʻia me Annexin V FITC a me PI
ʻIkepili Kaʻapuni Pūnaewele o ka Pūnaehana maʻi maʻi
ʻO ka pōʻaiapili pūnaewele a i ʻole ka pōʻaiapili pūnaewele ʻo ia ke ʻano o nā hanana i hana ʻia i loko o kahi keena e alakaʻi ana i kāna mahele a me ka hana hou ʻana o kāna DNA (DNA replication) e hana i ʻelua mau keena kaikamahine.I loko o nā pūnaewele me kahi nucleus, e like me nā eukaryotes, ua māhele ʻia ka pōʻaiapili cell i ʻekolu mau manawa: interphase, ka mitotic (M) phase, a me ka cytokinesis.ʻO ka propidium iodide (PI) he mea hoʻoheheʻe nuklea i hoʻohana pinepine ʻia e ana i ka pōʻaiapili kelepona.No ka mea ʻaʻole hiki i ka wai ke komo i nā sela ola, ua hoʻopaʻa ʻia nā cell me ka ethanol ma mua o ka hoʻopaʻa ʻana.Hoʻopaʻa ʻia nā cell a pau.Loaʻa i nā pūnaewele e hoʻomākaukau ana no ka māhele ʻana i ka nui o ka DNA a hōʻike i ka fluorescence hoʻonui proportionally.Hoʻohana ʻia nā ʻokoʻa i ka ikaika fluorescence e hoʻoholo i ka pākēneka o nā cell i kēlā me kēia māhele o ka pōʻai cell.Hiki iā Countstar ke hopu i ke kiʻi a e hōʻike ʻia nā hopena ma ka polokalamu FCS express.(Kiʻi 2)
Kiʻi 2: ʻO MCF-7 (A) a me 293T (B) i hoʻopaʻa ʻia me ke kelepona ʻike ʻana i ke kelepona me PI, ua hoʻoholo ʻia nā hopena e Countstar Rigel, a ua loiloi ʻia e FCS express.
ʻO ka hoʻoholo ʻana i ka hoʻololi ʻana o ka GFP ma ke kelepona
I ka wā o ka bioprocess, hoʻohana pinepine ʻia ʻo GFP e hui pū me ka protein recombinant ma ke ʻano he hōʻailona.E hoʻoholo i ka GFP fluorescent hiki ke hōʻike i ka hōʻike protein target.Hāʻawi ʻo Countstar Rigel i kahi hōʻike wikiwiki a maʻalahi no ka hoʻāʻo ʻana i ka transfection GFP a me ka hiki.Ua hoʻopaʻa ʻia nā kelepona me ka Propidium iodide (PI) a me Hoechst 33342 e wehewehe i ka heluna kanaka make a me ka heluna o nā cell cell.Hāʻawi ʻo Countstar Rigel i kahi ala wikiwiki a nui no ka loiloi ʻana i ka maikaʻi o ka hōʻike GFP a me ka hiki i ka manawa like.(Kiʻi 4)
Kiʻi 4: Aia nā pūnaewele me ka hoʻohana ʻana i ka Hoechst 33342 (uliuli) a hiki ke hoʻoholo maʻalahi i ka pākēneka o ka GFP e hōʻike ana i nā cell ('ōmaʻomaʻo).Hoʻopili ʻia nā cell nonviable me propidium iodide (PI; ʻulaʻula).
Ka Viability a me ka helu kelepona
ʻO ka helu AO/PI Dual-fluoresces ka ʻano assay i hoʻohana ʻia no ka ʻike ʻana i ka neʻe ʻana o ka cell, viability.Hoʻokaʻawale ʻia i ka helu kelepona a me ka helu kelepona mua e like me ke ʻano kelepona ʻokoʻa.Aia i loko o ka hoʻonā ka hui pū ʻana o ka ʻakika nucleic acid green-fluorescent, ka ʻalani acridine, a me ka stain nucleic acid redfluorescent, propidium iodide.ʻO ka propidium iodide he mea hoʻoheheʻe hoʻokaʻawale membrane e komo wale ana i nā keena me nā membrane i hoʻohālikelike ʻia aʻo ka ʻalani acridine e komo i nā cell āpau i kahi heluna.Ke loaʻa nā wai ʻelua i loko o ka nucleus, ʻo propidium iodide ka mea e hoʻemi ai i ka fluorescence ʻalani acridine ma o ka hoʻololi ʻana i ka ikehu resonance (FRET).ʻO ka hopena, ʻo nā pūnaewele nucleated me nā membrane paʻa i hoʻopaʻa i ka ʻōmaʻomaʻo ʻōmaʻomaʻo a helu ʻia he ola, ʻoiai nā cell nucleated me nā membrane i hoʻopaʻapaʻa ʻia wale nō ka ʻulaʻula fluorescent a helu ʻia he make ke hoʻohana ʻia ka ʻōnaehana Countstar Rigel.ʻAʻole ʻike ʻia nā mea non-nucleated e like me ke koko ʻulaʻula, platelets a me nā ʻōpala a nānā ʻole ʻia e ka polokalamu Countstar Rigel.(Kiʻi 5)
Kiʻi 5: Ua hoʻoikaika ʻo Countstar i kahi ʻano hana ʻulaʻula lua-fluorescence no ka maʻalahi a me ka pololei o ka hoʻoholo ʻana o ka PBMC a me ka hiki.Hiki ke nānā ʻia nā laʻana me ka AO/PI me ka Counstar Rigel
