Kusanthula ma leukocyte m'magazi athunthu ndi kuyesa kokhazikika mu labotale yachipatala kapena nkhokwe ya magazi.Kukhazikika ndi kuthekera kwa ma leukocyte ndizomwe zimafunikira pakuwongolera kusungidwa kwa magazi.Kupatula leukocyte, magazi athunthu amakhala ndi mapulateleti ambiri, maselo ofiira a magazi, kapena zinyalala zama cell, zomwe zimapangitsa kuti zikhale zosatheka kusanthula magazi athunthu mwachindunji pansi pa microscope kapena kauntala ya cell field.Njira zodziwika bwino zowerengera maselo oyera amwazi zimaphatikizapo njira ya RBC lysis, yomwe imatenga nthawi.
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).Zotsatira zake, ma cell a nucleated okhala ndi nembanemba osawoneka bwino amadetsa zobiriwira za fulorosenti ndipo amawerengedwa kuti ndi amoyo, pomwe ma cell a nucleated okhala ndi nembanemba osokonekera amangowononga zofiira za fulorosenti ndipo amawerengedwa ngati akufa akamagwiritsa ntchito Countstar® Rigel system.
Countstar Rigel ndi yankho loyenera pamayesero ovuta ambiri amtundu wa ma cell, omwe amatha kusanthula mwachangu maselo oyera amagazi athunthu.
Njira Yoyesera:
1.Tengani 20 µl ya magazi ndi kusungunula chitsanzocho mu 180 µl ya 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.Choose "AO/PI Viability" assay, ndiye Lowani Chitsanzo ID chitsanzo ichi.
7.Select Dilution ratio, Cell Type, dinani 'Thamangani' kuti muyambe kuyesa.
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 Image ya magazi athunthu
Pachithunzithunzi chowala chamagazi onse, ma WBC sawoneka pakati pa maselo ofiira a magazi.(Chithunzi 1)
Chithunzi 1 Chithunzi chowala chamagazi athunthu.
2. Fluorescence Chithunzi cha magazi athunthu
Utoto wa AO ndi PI onse ndi madontho a DNA mu phata la cell.Chifukwa chake, ma Platelets, maselo ofiira amwazi, kapena zinyalala zama cell sangathe kukhudza ndende ya leukocyte ndi zotsatira zake.Ma leukocyte amoyo (Obiriwira) ndi ma leukocyte akufa (Ofiira) amawonekera mosavuta pazithunzi za fluorescence.(Chithunzi 2)
Chithunzi 2 Fluorescence Zithunzi zamagazi athunthu.(A).Chithunzi cha AO Channel;(B) Chithunzi cha PI Channel;(C) Phatikizani zithunzi za AO ndi PI Channel.
3. Kukhazikika ndi kuthekera kwa leukocytes
Mapulogalamu a Countstar FL amawerengera okha ma cell a zigawo zitatu za zipinda ndikuwerengera mtengo wa chiwerengero chonse cha WBC cell count (1202), concentration (1.83 x 106 cells/ml), ndi % viability (82.04%).Zithunzi zonse zamagazi ndi deta zitha kutumizidwa kunja mosavuta ngati PDF, Image kapena Excel kuti muwunikenso kapena kusungitsa deta.
Chithunzi 3 Chithunzi chazithunzi cha Countstar Rigel Software
