Practical molecular biology PD Dr. Alexei Gratchev Prof. Dr. Julia Kzhyshkowska Prof. Dr. Wolfgang Kaminski

Course structure  08.10Plasmids, restriction enzymes, analytics  09.10Genomic DNA, RNA  10.10PCR, real-time (quantitative) PCR  11.10Protein analysis IHC  12.10Flow cytometry (FACS)

Nomenclature  FACS – Fluorescence Assisted Cell Sorting  FACS analysis  Flow cytometry  Flow cytofluorometry

Applications  Medicine  Immunophenotyping of blood cells  Diagnostic of various hematologic diseases  Transplantation  Research  Cell cycle analysis  Expression analysis  Phagocytosis, endocytosis  Cytokine production analysis

History  Cytophotometry. UMSP-1 (Zeiss) 5-10 min/cell  1969 Pulse cytophotometry. ICP-11 (Phywe, Göttingen) (in 1976 purchased by Ortho-diagnostics (USA) and later disappeared from the market)  Mercury lamp excitation ( nm)  2 fluorescent parameters PMT detectors (no scatter detectors)  >1000 cells/sec  1970 Cytofluorograph (Ortho-Diagnostics)  Argon laser (488 nm) as a light source  1976 Dual laser instrument (DKFZ, Heidelberg)  1986 First sorter (PARTEC, Münster)  Cell sizing option (Bruker-Odam, France)

Principle of flow cytometry Hydrodynamic focusing of the cells in the focused laser beam

Optical scheme of a flow cytometer

4 colour flow cytometer

Optical system of BD FACSAria II

2 laser flow cell

Fluorophores

Parameters collected  FSC – Forward scatter (correlates with the cell size)  SSC –Sideward scatter (correlates with the cell granulation)  FL – Fluorescence signal (min 3, max 12 channels)  Concentration of cells (only with some flow cytometers)  Size of the cell (only with some flow cytometers)

Flow cytometry results

Compensation problem

4 colour compensation

Compensation  Measure cells labelled with single antibody  Determine the percentage of the signal in wrong detector  Generate compensation matrix  Modern cytometers perform compensation automatically

Experiment planning  Antibody has to be highly specific  Antibody has to be tittered  Select correct fluorophores (low expression – bright fluorescence, high expression – weak fluorescence)  Think about the abundance of the cell population you want to analyse

Controls  Non stained cells  Cells stained with single antibodies/dyes (for compensation purpose)  Cells stained with unspecific isotype control – unspecific antibodies of the same isotype as your test antibodies, labeled with the same fluorophore with the same efficiency

Cell staining  Prepare cell suspension  Add antibody  Incubate  Wash  Measure

Experiment today a)2 µl CD14 FITC Ab b)2 µl CD16 APC Ab c)2 µl CD14 FITC Ab + CD16 APC Ab d)2 µl Isotype FITC e)2 µl Isotype APC f)2 µl Isotype FITC + 2 µl Isotype APC g)Non-labelled cells

Questions?