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Durchflußzytometrie und FACS-Sortierung für Einsteiger und Fortgeschrittene

Durchflußzytometrie und FACS-Sortierung für Einsteiger und Fortgeschrittene. Skript: http://www.biologie.uni-erlangen.de/genetik/teach_scripts.deu.php. Injector Tip. Sheath fluid. Fluorescence. signals. Focused laser. beam. Flow Cell. Laser. FALS Sensor.

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Durchflußzytometrie und FACS-Sortierung für Einsteiger und Fortgeschrittene

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  1. Durchflußzytometrie undFACS-Sortierung fürEinsteiger undFortgeschrittene Skript: http://www.biologie.uni-erlangen.de/genetik/teach_scripts.deu.php

  2. Injector Tip Sheath fluid Fluorescence signals Focused laser beam Flow Cell

  3. Laser FALS Sensor Forward Angle Light Scatter

  4. Laser FALS Sensor 90LS Sensor 90 Degree Light Scatter

  5. 1 Erythrocytes 2 small Lymphocytes 4 3 large Lymphocytes „Blasts“ 4 Granulocytes + Macrophages 1 3 2 FSC / SSC of bone marrow

  6. FALS Sensor Fluorescence Fluorescence detector (PMT3, PMT4 etc.) Fluorescence Detectors Laser

  7. positive Frequency of events Signal intensity (fluorescence) Histograms 32%

  8. almost perfect negative populationslightly shifted Frequency of events unstained Overlay stained Signal intensity (fluorescence) Controls

  9. Autofluorescence Analysed with Flowjo (http://www.flowjo.com)(Windows and Mac, can read all file types)

  10. Titrieren der Antikörper

  11. Titrieren der Antikörper

  12. Take home messageAntibody controls • Best negative control: cellular control:-> specific antibody on cells that do not express the antigen • Isotype controls are problematic because:the ratio of fluorochrome to antibody always varies between different antibody conjugate • Background autofluorescence as well as unspecific staining is different for different cell types (Lymphocytes versus macrophages; large cells versus small cells)

  13. Flow Cytometry Optics PMT 4 PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser

  14. 632.5 nm (HeNe) Excitation Emisson Allophycocyanin (APC) Protein 300 nm 400 nm 500 nm 600 nm 700 nm

  15. 350 457 488 514 610 632 300 nm 400 nm 500 nm 600 nm 700 nm Common Laser Lines PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid

  16. TWO COLOR IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin

  17. Why you have to compensate A: FL-1 signal in detector for FL2 (strong for FITC into PE) B: FL2 signal in detector for FL1 (weak for PE into FITC)

  18. compensated 4 4 10 10 3 3 10 10 2 2 10 10 1 1 10 10 0 0 10 10 0 1 3 2 4 0 1 3 2 4 10 10 10 10 10 10 10 10 10 10 Kompensation uncompensated fluorescence 2 fluorescence 1

  19. uncompensated partially compensated fluorescence 2 4 4 10 10 3 3 10 10 2 2 10 10 fully compensated 1 1 10 10 0 0 10 10 0 1 3 2 4 0 1 3 2 4 10 10 10 10 10 10 10 10 10 10 4 10 3 10 2 10 fluorescence 1 1 10 0 10 0 1 3 2 4 10 10 10 10 10 Die richtige Kompensation ist abhängig von der Signalstärke!!

  20. Take home messageCompensation controls • All single colour stainings (i.e. for FITC/PE staining):single FITC labeled + single PE labeledfor 4-colour experiment: 4 controls • Use markers where at least 5 - 10 % positive cells are expected • Use strong signals for compensation • For tandem dyes (PE-Cy5 etc...): the experimental antibody conjugate has to be used for compensation control (different conjugate ratios! And even Lot to Lot variation!) • Prepare a lot of cells (and volume) for the compensation controls as it take time to set up the machine • Some new programms (and FACS machines work with software compensation

  21. 4 4 10 10 3 3 10 10 2 2 10 10 1 1 10 10 0 0 10 10 0 1 3 2 4 0 1 3 2 4 10 10 10 10 10 10 10 10 10 10 PI gating für lebende Zellen • Tote Zellen besitzen hohe Autofluoreszenz • Tote Zellen binden unspezifisch an Antikörper VIABLE CELLS ALL CELLS A B dead cells PE-LAMBDA FL-KAPPA

  22. PI gating für lebende Zellen Propidiumiodid 1 mg/ml Zellen auf Eis PI möglichst erst kurz vor derMessung zugeben PI FL3 gate forward scatter

  23. How many colours? 1 Argon Laser

  24. How many colours? 1 HeNe Laser

  25. How many colours? 1 UV Laser

  26. How many colours? 1 Argon, 1 HeNe Laser

  27. How many colours? maybe 10.....

  28. + Fluorescence Activated Cell Sorting FALS Sensor 488 nm laser Fluorescence detector - Charged Plates Single cells sorted into test tubes

  29. Zellsortierung am MoFlo • Max. Sortiergeschwindigkeit 20.000 Zellen / SekundeBeispiel: 3 Maus-Milzen ca. 200 Mio Zellen-> ca. 4 Std. • Voranreicherung z.B. durch MACS ermöglicht noch mehr Durchsatz • Verschiedene „Purity Modes“: single cell, enrich, purewesentlicher Unterschied: Geschwindigkeit versus abort rate => Ausbeute • Einzelzell-Sortierung in Zellkulturplatten möglich • Sterilität? • Sensitivität niedriger als am FACScalibur (Quarzküvette versus Luft) • Filtern der Zellsuspension sehr wichtig: Nozzledurchmesser 70 mM (Shapiros Law!) • Kühlung ist möglich und wichtig für längere Sorts von lebenden Zellen (Capping) • In die Auffangröhrchen Medium / FCS vorlegen! • Nach dem Sort länger und mit höherer Drehzahl abzentrifugierenKontrollversuch: 5 min 1200 U/min 50% Ausbeute, 10 min 2000 U/min -> mehr als 90% Ausbeute entspr. Zähler am MoFlo

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