A na-ahazi mkpụrụ ndụ mononuclear ọbara dị n'akụkụ (PBMCs) iji kewapụ na ọbara dum site na njupụta gradient centrifugation.Selụ ndị ahụ nwere lymphocytes (sel T, mkpụrụ ndụ B, mkpụrụ ndụ NK) na monocytes, nke a na-ejikarị eme ihe na ngalaba nke immunology, ọgwụgwọ cell, ọrịa na-efe efe na mmepe ọgwụ mgbochi.Nleba anya na nyochaa ike na itinye uche nke PBMC dị oké mkpa maka ụlọ nyocha ụlọ ọgwụ, nchọpụta sayensị ahụike na mmepụta cell dịghịzi.
Fig 1. PBMC dịpụrụ adịpụ site na ọbara ọhụrụ na Density gradient centrifugation
AOPI Dual-fluoresces agụta bụ ụdị nyocha eji achọpụta mkpokọta cell na ịdị adị.Ihe ngwọta bụ nchikota nke acridine oroma (acha ndụ ndụ-fluorescent nucleic acid unyi) na propidium iodide (red-fluorescent nucleic acid ntụpọ).Propidium iodide (PI) bụ ụcha mwepu nke akpụkpọ ahụ nke na-abanye naanị n'ime sel nwere akpụkpọ ahụ mebiri emebi, ebe oroma acridine na-enwe ike ịbanye n'ime sel niile na ọnụ ọgụgụ mmadụ.Mgbe agbaji abụọ dị na nucleus, propidium iodide na-eme ka mbelata nke acridine oroma fluorescence site na fluorescence resonance energy transfer (FRET).N'ihi nke a, mkpụrụ ndụ nucleated nwere akpụkpọ ahụ na-adịghị emebi emebi na-acha akwụkwọ ndụ akwụkwọ ndụ na-acha akwụkwọ ndụ, ebe a na-agụkwa ya dị ka ndị dị ndụ, ebe sel ndị nwere nsogbu mebie na-acha uhie uhie na-acha uhie uhie na-agụkwa dị ka ndị nwụrụ anwụ mgbe a na-eji usoro Countstar® FL.Ihe anaghị emebi emebi dị ka mkpụrụ ndụ ọbara uhie, platelet na irighiri ihe anaghị apụta ma Countstar® FL na-eleghara ya anya.
Usoro nnwale:
1.Dilute PBMC sample n'ime 5 iche iche iche iche na PBS;
2. Tinye 12µl AO / PI ngwọta n'ime 12µl sample, jiri nwayọọ gwakọta ya na pipette;
3.Draw 20µl ngwakọta n'ime ụlọ slide;
4. Kwe ka sel ahụ nọrọ n'ime ụlọ maka gburugburu 1 nkeji;
5.Insect slide ahụ n'ime ngwá ọrụ Countstar FL;
6.Họrọ nyocha "AO/PI Viability", wee nwalee site na Countstar FL.
Akpachara anya: AO na PI nwere ike ibute ọrịa carcinogen.A na-atụ aro ka onye na-ahụ maka ọrụ yikwasị ngwa nchedo onwe onye (PPE) ka ịzenarị akpụkpọ anụ na anya ozugbo.
Nsonaazụ:
1.Bright Field na Fluorescence oyiyi nke PBMC
AO na PI agba bụ ma ntụpọ DNA na cell nucleus nke sel.Ya mere, Platelet, mkpụrụ ndụ ọbara uhie, ma ọ bụ irighiri ihe cellular enweghị ike imetụta ntinye uche na nsonaazụ ndụ PBMC.Enwere ike ịwapụta mkpụrụ ndụ dị ndụ, mkpụrụ ndụ nwụrụ anwụ na irighiri ihe n'ụzọ dị mfe na onyonyo nke Countstar FL mepụtara (Njirimara 1).
Onyonyo 2. Igwe na-egbuke egbuke na ihe oyiyi Fluorescence nke PBMC
2.Concentration na Viability nke PBMC
Ejiri PBS gbazere ihe nlele PBMC na 2, 4, 8 na 16, mgbe ahụ, etinyere ihe nlele ahụ na ngwakọta AO/PI wee nyochaa ya site na Countstar FL n'otu n'otu.E gosipụtara nsonaazụ nke itinye uche na ike nke PBMC dị ka ọnụ ọgụgụ dị n'okpuru:
