Zotumphukira zamagazi a mononuclear cell (PBMCs) nthawi zambiri amasinthidwa kuti alekanitse ndi magazi athunthu ndi kachulukidwe kachulukidwe ka centrifugation.Maselo amenewa ali ndi ma lymphocytes (maselo a T, maselo a B, NK maselo) ndi ma monocytes, omwe amagwiritsidwa ntchito kwambiri pamagulu a immunology, chithandizo cha maselo, matenda opatsirana ndi katemera.Kuwunika ndikuwunika momwe PBMC imagwirira ntchito ndikofunikira pama labotale azachipatala, kafukufuku woyambira wa sayansi ya zamankhwala komanso kupanga maselo a chitetezo chamthupi.
Chithunzi 1. Isolated PBMC kuchokera kumagazi atsopano okhala ndi Density gradient centrifugation
Kuwerengera kwa AOPI Dual-fluoresces ndi mtundu woyeserera womwe umagwiritsidwa ntchito pozindikira kuchuluka kwa ma cell ndi kuthekera kwake.Njira yothetsera vutoli ndi kuphatikiza acridine lalanje (dontho la green-fluorescent nucleic acid) ndi propidium iodide (dontho lofiira la fulorosenti la nucleic acid).Propidium iodide (PI) ndi utoto wochotsa nembanemba womwe umangolowa m'maselo okhala ndi nembanemba wosokonekera, pomwe acridine orange amatha kulowa m'maselo onse mwa anthu.Mitundu yonse iwiri ikakhala mu phata, propidium iodide imayambitsa kuchepa kwa acridine orange fluorescence ndi fluorescence resonance energy transfer (FRET).Chotsatira chake, maselo a nucleated omwe ali ndi nembanemba osasunthika amadetsa zobiriwira za fulorosenti ndipo amawerengedwa kuti ndi amoyo, pamene maselo a nucleated omwe ali ndi nembanemba yowonongeka amangowononga zofiira za fulorosenti ndipo amawerengedwa ngati akufa pogwiritsa ntchito Countstar® FL system.Zinthu zopanda ma nucleated monga maselo ofiira a magazi, mapulateleti ndi zinyalala sizimasungunuka ndipo zimanyalanyazidwa ndi pulogalamu ya Countstar® FL.
Njira Yoyesera:
1.Dilute chitsanzo cha PBMC mumagulu osiyanasiyana a 5 ndi PBS;
2.Onjezani yankho la 12µl AO/PI mu chitsanzo cha 12µl, chosakanizidwa bwino ndi pipette;
3. Jambulani kusakaniza kwa 20µl mu slide ya chipinda;
4.Lolani ma cell kuti akhazikike mchipindamo kwa mphindi imodzi;
5.Isect slide mu chida cha Countstar FL;
6.Sankhani kuyesa kwa "AO/PI Viability", kenako yesani ndi Countstar FL.
Chenjezo: AO ndi PI ndizomwe zimayambitsa khansa.Ndikofunikira kuti wogwiritsa ntchito azivala zida zodzitetezera (PPE) kuti apewe kukhudzana mwachindunji ndi khungu ndi maso.
Zotsatira:
1.Bright Field ndi zithunzi za Fluorescence za PBMC
Utoto wa AO ndi PI onse ndi madontho a DNA mu phata la cell.Chifukwa chake, ma Platelet, maselo ofiira amwazi, kapena zinyalala zama cell sangathe kukhudza kukhazikika kwa ma PBMC ndi zotsatira zake.Maselo amoyo, maselo akufa ndi zinyalala amatha kusiyanitsa mosavuta pazithunzi zopangidwa ndi Countstar FL (Chithunzi 1).
Chithunzi 2. Zithunzi za Bright Field ndi Fluorescence za PBMC
2.Kukhazikika ndi Kutheka kwa PBMC
Zitsanzo za PBMC zidachepetsedwa mu 2, 4, 8 ndi 16 nthawi ndi PBS, ndiye zitsanzozo zidakulungidwa ndi utoto wa AO / PI ndikuwunikidwa ndi Countstar FL motsatana.Zotsatira za ndende komanso kuthekera kwa PBMC zikuwonetsedwa pansipa:
