Hoʻomaʻamaʻa pinepine ʻia nā cell mononuclear blood peripheral (PBMCs) e hoʻokaʻawale mai ke koko holoʻokoʻa e ka centrifugation gradient density.ʻO kēlā mau pūnaewele he lymphocytes (nā pūnaewele T, nā pūnaewele B, nā pūnaewele NK) a me nā monocytes, i hoʻohana mauʻia i ke kahua o ka immunology, cell therapy, infectious disease and vaccine development.He mea koʻikoʻi ka nānā ʻana a me ka nānā ʻana i ka viability a me ka noʻonoʻo ʻana o ka PBMC no nā laboratories clinical, ka noiʻi ʻepekema lapaʻau kumu a me ka hana ʻana o nā cell immune.
Fig 1. Isolated PBMC mai ke koko hou me Density gradient centrifugation
ʻO ka helu AOPI Dual-fluoresces ka ʻano assay i hoʻohana ʻia no ka ʻike ʻana i ka neʻe ʻana o ke kelepona a me ka hiki.ʻO ka hoʻonā ʻana he hui pū ʻana o ka ʻalani acridine (ka ʻakika nucleic acid green-fluorescent) a me ka propidium iodide (ka ʻulaʻula nucleic acid stain).ʻO ka propidium iodide (PI) he mea hoʻoheheʻe hoʻokaʻawale membrane e komo wale i nā pūnaewele me nā membrane i hoʻopaʻa ʻia, ʻoiai hiki i ka ʻalani acridine ke 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, hoʻopaʻa ʻia nā sela nucleated me nā membrane paʻa i ka ʻōmaʻomaʻo ʻōmaʻomaʻo a helu ʻia he ola, akā ʻo nā cell nucleated me nā membrane i hoʻopaʻa ʻia e hoʻopaʻa wale i ka ʻulaʻula fluorescent a helu ʻia he make ke hoʻohana i ka ʻōnaehana Countstar® FL.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® FL.
Kaʻina hana hoʻokolohua:
1. E hoʻoheheʻe i ka hāpana PBMC i loko o 5 mau ʻokoʻa ʻokoʻa me PBS;
2. Hoʻohui i ka 12µl AO / PI solution i loko o ka hāpana 12µl, hui mālie me ka pipette;
3. E huki i ka hui ʻana o 20µl i loko o ke keʻena slide;
4. E hoʻokuʻu i nā pūnaewele e noho i loko o ke keʻena no kahi o 1 minuke;
5. E ʻoki i ka paheʻe i loko o ka mea kani Countstar FL;
6. E koho i ka "AO/PI Viability" assay, a laila ho'āʻo e Countstar FL.
E akahele: ʻO AO a me PI he mea make kino.Manaʻo ʻia e hoʻohana ka mea hoʻohana i nā lako pale pilikino (PPE) e pale aku i ka pili pono ʻana me ka ʻili a me nā maka.
Ka hopena:
1.Bright Field a me nā kiʻi Fluorescence o ka PBMC
ʻO ka dye AO a me ka PI he mau kiko DNA ʻelua i loko o ka nucleus cell of cell.No laila, ʻaʻole hiki i nā Platelets, nā ʻulaʻula ʻulaʻula, a i ʻole nā ʻōpala cellular ke hoʻopilikia i ka manaʻo o ka PBMC a me ka hopena viability.Hiki ke ʻike maʻalahi ʻia nā mea ola, nā mea make a me nā ʻōpala ma muli o nā kiʻi i hana ʻia e Countstar FL (Figure 1).
Kiʻi 2. Nā kiʻi Māmā a me Fluorescence o ka PBMC
2.Concentration a me ka Viability o PBMC
Ua hoʻoheheʻe ʻia nā laʻana PBMC i 2, 4, 8 a me 16 mau manawa me ka PBS, a laila ua hoʻomoʻi ʻia kēlā mau laʻana me kahi hui wai AO/PI a nānā ʻia e Countstar FL.Hōʻike ʻia ka hopena o ka noʻonoʻo a me ka hiki ʻana o ka PBMC e like me ke kiʻi ma lalo nei:
