Peripheral ntshav mononuclear hlwb (PBMCs) feem ntau ua tiav los ntawm cov ntshav tag nrho los ntawm qhov ntom ntom gradient centrifugation.Cov hlwb no muaj cov lymphocytes (T hlwb, B hlwb, NK hlwb) thiab monocytes, feem ntau siv hauv kev tiv thaiv kab mob, kev kho cell, kab mob sib kis thiab kev tsim tshuaj tiv thaiv.Kev saib xyuas thiab tshuaj xyuas kev muaj peev xwm thiab kev xav tau ntawm PBMC yog qhov tseem ceeb rau cov chaw kuaj mob, kev tshawb fawb txog kev kho mob yooj yim thiab kev tiv thaiv kab mob.
Daim duab 1. cais PBMC los ntawm cov ntshav tshiab nrog Kev Ntxig gradient centrifugation
AOPI Dual-fluoresces suav yog hom tshuaj ntsuam xyuas siv los kuaj xyuas cov cell concentration thiab muaj sia nyob.Cov tshuaj yog ua ke ntawm acridine txiv kab ntxwv (cov ntsuab-fluorescent nucleic acid stain) thiab propidium iodide (liab-fluorescent nucleic acid stain).Propidium iodide (PI) yog ib daim nyias nyias nyias dye uas tsuas yog nkag mus rau hauv cov hlwb nrog kev cuam tshuam daim nyias nyias, thaum acridine txiv kab ntxwv muaj peev xwm nkag mus rau tag nrho cov hlwb hauv cov pejxeem.Thaum ob lub dyes muaj nyob rau hauv lub nucleus, propidium iodide ua rau txo qis hauv acridine txiv kab ntxwv fluorescence los ntawm fluorescence resonance zog hloov (FRET).Raws li qhov tshwm sim, cov cell nucleated nrog cov kab mob tsis zoo stain fluorescent ntsuab thiab suav tias yog nyob, thaum cov cell nucleated nrog cov kab mob cuam tshuam tsuas yog stain fluorescent liab thiab suav tias tuag thaum siv Countstar® FL system.Cov khoom tsis muaj nucleated xws li cov qe ntshav liab, platelets thiab cov khib nyiab tsis muaj fluoresce thiab tsis quav ntsej los ntawm Countstar® FL software.
Cov txheej txheem sim:
1.Dilute tus qauv PBMC rau hauv 5 qhov sib txawv nrog PBS;
2.Ntxiv 12µl AO/PI tov rau hauv 12µl qauv, maj mam tov nrog pipette;
3.Draw 20µl sib tov rau hauv chamber swb;
4.Cia cov hlwb los nyob hauv lub chamber li ntawm 1 feeb;
5.Insect tus swb rau hauv Countstar FL ntsuas;
6.Xaiv qhov kev ntsuam xyuas "AO/PI Viability", tom qab ntawd sim los ntawm Countstar FL.
Ceeb Toom: AO thiab PI yog cov muaj peev xwm carcinogen.Nws raug nquahu kom tus neeg teb xov tooj hnav cov cuab yeej tiv thaiv tus kheej (PPE) kom tsis txhob cuam tshuam ncaj qha rau ntawm daim tawv nqaij thiab qhov muag.
Qhov tshwm sim:
1.Ntxim Field thiab Fluorescence duab ntawm PBMC
Cov AO thiab PI dye yog ob qho tib si stains DNA hauv cov cell nucleus ntawm cov hlwb.Yog li ntawd, Platelets, cov qe ntshav liab, los yog cov khib nyiab ntawm tes tsis tuaj yeem cuam tshuam rau PBMCs concentration thiab qhov ua tau zoo.Cov hlwb nyob, cov hlwb tuag thiab cov khib nyiab tuaj yeem yooj yim sib txawv ntawm cov duab tsim los ntawm Countstar FL (Daim duab 1).
Daim duab 2 .Zaj teb thiab cov duab fluorescence ntawm PBMC
2.Concentration thiab Viability ntawm PBMC
Cov qauv PBMC tau diluted hauv 2, 4, 8 thiab 16 zaug nrog PBS, tom qab ntawd cov qauv no tau tsim nrog AO / PI dye sib tov thiab tshuaj xyuas los ntawm Countstar FL feem.Qhov tshwm sim ntawm kev concentration thiab kev muaj peev xwm ntawm PBMC yog pom raws li hauv qab no:
