Aplikacije pretočne citometrije
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1 Aplikacije pretočne citometrije April, 2008
2 Flow cytometry-pretočna citometrija - Merjenje karakteristik celic v tekočini - Podobnost s fluorescenčno mikroskopijo - Analiza kompleksnih celičnih populacij - Sortiranje
3 Altra
4 Princip pretočne citometrije
5 Merilna komora - Flow Cell Izotonična tekočina Fluorescenčni signali Laserski žarek Purdue University Cytometry Laboratories
6 Forward Angle Light Scatter Laser FALS Sensor Purdue University Cytometry Laboratories
7 Side Scatter Laser FALS Sensor 90LS Sensor Purdue University Cytometry Laboratories
8 Fluorescence Detectors Laser FALS Sensor Freq Fluorescence Fluorescence detector (PMT3, PMT4 etc.) Purdue University Cytometry Laboratories
9 Aplikacije pretočne citometrije
10 Primeri uporabe pretočne citometrije - sledenje proteinov imunofluorescenca - analiza krvi - sledenje apoptoze - analiza celic iz tkiv - analiza celičnega cikla - sortiranje
11 Sledenje proteinov z imunofluorescenco - ugotavljanje specifičnih epitopov na proteinu (npr. aktivost kinaz z uporabo fosfo-specifičnih protiteles)
12 Interakcije med proteini
13 Analiza krvnih celic Scale Forward scatter Neutrofilci Limfociti Monociti Side Scatter Purdue University Cytometry Laboratories
14 Določanje koncentracije neutrofilcev v krvi Neutrofilci so označeni s specifičnimi primarnimi protitelesi in sek. protitelesi, ki imajo vezan FITC. (undergoing review - Škrajnar Š, Lasnik MA, and Zavec AB: A flow cytometric method for rapid determination of blood neutrophil concentration in rats.)
15 Apoptoza, merjena z Aneksinom V in PI V601_ PI_Alexa DOX_V601_PI_Alexa (Smilovič V, Zavec AB)
16 Apoptoza, merjena s spremembo membranskega potenciala mitohondrijev DePsipher Kit: detekcija membranskega potenciala M (kationsko barvilo prehaja v M in sveti rdeče kot multimera; če M potencial pade, barvilo ostane v citoplazmi v monomerni zeleni obliki. Odrasle živali Mladički (Erman A, Zavec AB)
17 Količina mitohondrijev v regeneracijskem tkivu Analiza iz tkivnih c.: tripsin, kolagenaza MitoTracker Green: se veže na M v živih in fiksiranih c. (Goršič M, Zavec AB)
18 Analiza celičnega cikla G 2 M G 0 Analiza DNA G 1 s C o u n t G 0 G 1 s G 2 M N 4N DNA content Purdue University Cytometry Laboratories
19 Sledenje poteka mejotične replikacije DNA-PI (Zavec AB, Lešnik U, Komel R, and Comino A: The Saccharomyces cerevisiae gene ECM11 is a positive effecter of meiosis; FEMS Microbiol Lett. 2004)
20 Celični cikel in poliploidnost DNA-PI Wt Small Big (undergoing review - Nikitin D, Tosato V, Zavec AB, and Bruschi CV: Cellular and molecular effects of chromosome translocations in Saccharomyces cerevisiae) Val
21 Sortiranje Fluorescence Activated Cell Sorting (FACS) 488 nm laser FALS senzor detektorji fluorescence nabiti plošči V - + posamezne celice sortirane v kiveti Purdue University Cytometry Laboratories
22 Sortiranje EYFP-aktin (Hrovat A, Zavec AB)
23 Sortiranje Celična linija CHO, transformirana z receptorjem NK1-R(HA), prim.ab rat anti-ha, sekab anti-rat PE. (Hrovat A, Zavec AB)
24 Prednosti pretočne citometrije visoka občutljivost metode izjemno hitro zaznavanje več parametrov hkrati: velikost, granuliranost, fluorescenca: velika in nesubjektivna statistika največkrat gre za meritve na živih celicah zaznavanje lastnosti posameznih celic: identifikacija celičnih podskupin v kompleksni populaciji
25 Aplikacije pretočne citometrije April, 2008
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