Vrednotenje celične linije PC-12 s pretočno citometrijo s sočasnim zajemanjem slike

Lipovnik, Anja (author), Doljak, Bojan (mentor)

V diplomskem delu smo vrednotili celično linijo PC-12, ki predstavlja dober nevrološki model za raziskavo nevrodegenerativnih bolezni. Analize smo opravili s pretočno citometrijo s sočasnim zajemanjem slike. Slikovni citometer ImageStream združuje kovencionalno pretočno citometrijo in sodobno fluorescenčno mikroskopijo. Omogoča multiparametrične analize, s katerimi lahko hitro in učinkovito karakteriziramo lastnosti posameznih celic, kot so velikost, oblika, tekstura, granuliranost in intenzivnost fluorescence. Celice smo označili s fluorofornimi označevalci (DAPI, Alexa Fluor 488, Alexa Fluor 633, TRITC) neposredno, s faloidinom ali posredno s protitelesi. S pomočjo programske opreme IDEAS preučili delež živih celic PC-12 ter distribucijo aktina, cistatina C in katepsina X. S statistično analizo (določitev vrednost RD) smo na podlagi fenotipskih značilnosti celic ovrednotili subpopulacije in posamezne celice. Intenziteto fluorescence smo določili pri permeabiliziranih in nepermeabiliziranih celicah. Višje vrednosti intenzitete signala smo zaznali pri permeabiliziranih celicah, saj označena protitelesa lahko vstopijo v celice le, če je njihova membrana permeabilizirana.

• Language: Slovenian
• Keywords: pretočna citometrija, sočasno zajemanje slike, PC-12, fluorescenca
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-109899
• Publication date in RUL: 10.09.2019

Secondary language

• Language: English
• Title: Evaluating of PC-12 cell line using imaging flow cytometry

Abstract

In our thesis, we studied the PC-12 cell line, which represents a good neurological model for the research of neurodegenerative diseases. Analyses were performed using imaging flow cytometry. The ImageStream imaging cytometer combines conventional flow cytometry with modern fluorescence microscopy and allows multiparametric analyses of cell features, such as shape, texture, size, granularity and fluorescence intensity. Cells were labelled with fluorophores (DAPI, Alexa Fluor 488, Alexa Fluor 633, TRITC) directly, with phalloidin or indirectly with antibodies. Using the IDEAS software, we determined the portion of live cells and the distribution of actin, cystatin C and cathepsin X. With statistical analysis (determination of RD value) of phenotypic cell characteristics we studied subpopulations and individual cells. The fluorescence intensity was measured on permeabilised and non permeabilised cells. Higher signal strength was detected in permeabilised cells, as membrane permeabilisation allows labelled antibodies to enter into the cells.

• Keywords: Flow cytometry, simultaneous imaging, PC-12, fluorescence