Practical molecular
biology
PD Dr. Alexei Gratchev
Prof. Dr. Julia Kzhyshkowska
Prof. Dr. Wolfgang Kaminski
Course structure
08.10 Plasmids, restriction enzymes,
analytics
 09.10
Genomic DNA, RNA
 10.10
PCR, real-time (quantitative) PCR
 11.10
Protein analysis IHC
 12.10
Flow 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


1950-1960 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)




1970 Cytofluorograph (Ortho-Diagnostics)




Mercury lamp excitation (480-500nm)
2 fluorescent parameters PMT detectors (no scatter detectors)
>1000 cells/sec
Argon laser (488 nm) as a light source
1976 Dual laser instrument (DKFZ, Heidelberg)
1986 First sorter (PARTEC, Münster)
1990-1993 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
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)
b)
c)
d)
e)
f)
2 µl CD14 FITC Ab
2 µl CD16 APC Ab
2 µl CD14 FITC Ab + CD16 APC Ab
2 µl Isotype FITC
2 µl Isotype APC
2 µl Isotype FITC + 2 µl Isotype APC
g) Non-labelled cells
Questions?