Kwayoyin halittar jini guda ɗaya (PBMCs) galibi ana sarrafa su don raba su da duka jini ta hanyar haɓakar gradient mai yawa.Waɗancan ƙwayoyin sun ƙunshi lymphocytes (kwayoyin T, ƙwayoyin B, ƙwayoyin NK) da monocytes, waɗanda aka saba amfani da su a fagen ilimin rigakafi, jiyya na ƙwayoyin cuta, cututtukan cututtuka da haɓaka rigakafin rigakafi.Kulawa da yin nazari akan iyawa da tattarawar PBMC yana da mahimmanci ga dakunan gwaje-gwaje na asibiti, binciken kimiyyar likitanci na asali da samar da ƙwayoyin rigakafi.
Hoto 1. Keɓaɓɓen PBMC daga sabobin jini tare da Maƙarƙashiya gradient centrifugation
AOPI Dual-fluoresces ƙidayar ita ce nau'in tantancewar da ake amfani da ita don gano tattarawar tantanin halitta da yuwuwa.Maganin shine hadewar acridine orange (koren-fluorescent nucleic acid tabo) da kuma propidium iodide (tabon nucleic acid ja-fluorescent).Propidium iodide (PI) wani launi ne na keɓan membrane wanda kawai ke shiga sel tare da membranes masu rikitarwa, yayin da acridine orange yana iya shiga duk sel a cikin yawan jama'a.Lokacin da rinannun biyu ke kasancewa a cikin tsakiya, propidium iodide yana haifar da raguwar acridine orange fluorescence ta hanyar canja wurin makamashi mai haske (FRET).Sakamakon haka, ƙwayoyin sel waɗanda ke da matattun membranes suna lalata kore mai kyalli kuma ana ƙidaya su a matsayin masu rai, yayin da ƙwayoyin sel waɗanda ke da lalatawar membranes kawai suna lalata ja kuma ana ƙidaya su a matsayin matattu lokacin amfani da tsarin Countstar® FL.Abubuwan da ba su da ƙwayar cuta kamar ƙwayoyin jajayen jini, platelets da tarkace ba sa fitowa fili kuma software na Countstar® FL sun yi watsi da su.
Tsarin Gwaji:
1.Dilute samfurin PBMC a cikin nau'i daban-daban na 5 tare da PBS;
2. Ƙara 12µl AO / PI bayani a cikin samfurin 12µl, a hankali gauraye da pipette;
3. Zana 20µl cakuda cikin ɗakin zamewa;
4.Ba da izinin sel su zauna a cikin ɗakin don kimanin minti 1;
5.Insect da zamewar cikin Countstar FL kayan aiki;
6.Zaɓi gwajin "AO/PI Viability", sannan gwada ta Countstar FL.
Tsanaki: AO da PI ne mai yuwuwar cutar kansa.Ana ba da shawarar cewa ma'aikacin ya sa kayan kariya na sirri (PPE) don guje wa haɗuwa da fata da idanu kai tsaye.
Sakamako:
1.Bright Field da Fluorescence hotuna na PBMC
Rini na AO da PI duka suna tabo DNA a cikin tsakiya na sel.Saboda haka, Platelets, jajayen ƙwayoyin jini, ko tarkace ta salon salula ba su iya shafar tattarawar PBMCs da sakamako mai yiwuwa.Rayayyun sel, matattun ƙwayoyin cuta da tarkace ana iya bambanta su cikin sauƙi akan hotunan da Countstar FL ya haifar (Hoto 1).
Hoto 2. Hotunan Filin Haske da Hasken Haske na PBMC
2.Tattaunawa da Rigakafin PBMC
An narkar da samfuran PBMC a cikin 2, 4, 8 da 16 sau tare da PBS, sannan waɗannan samfuran an haɗa su tare da cakuda rini na AO/PI kuma Countstar FL sun bincika su bi da bi.An nuna sakamakon maida hankali da yiwuwar PBMC kamar yadda aka nuna a ƙasa:
