Extensive service features and technological innovations allow state-of-the art cell analysis and cell sorting.

The Flow Cytometry Core Facility (FCCF) is located at the DRFZ since the year 2000. It originated from the cooperation between the DRFZ, the Charite-Universitätsmedizin Berlin and the Max Planck Institute for Infection Biology.

The FCCF comprises of unique technical equipment. Scientists are provided the opportunity to conduct flow cytometric cell sorting and cell analyses.

The facility operates four cell sorters and tree cell analyzers, which are state-of-the-art instruments. The facility staff offers expertise based on a 12 year experience in the area of flow cytometry. It assists users offering competent consultation in the planning and realization of their experiments.

Methods and range of application of Flow Cytometry

Flow cytometry is an analytical method used for the quantitative analysis of physical, biochemical/cell biological and immunogenetic parameters of cells. On the basis of these parameters various cell features, such as viability, quantification of antigens, phenotype, and cell cycle can be determined. The flow cytometers available at the FCCF allow the simultaneous measurement of up to 15 parameters of cells. Cells can be measured and analyzed at an extremely rapid rate of up to 20,000 events per second. By this means, cell subpopulations cannot only be analyzed; they can also be isolated and grown, or used in genetic experiments. This is achieved by sorting a defined number of cells into tubes or on special cell culture plates. This procedure even makes the isola-tion of single cells possible.

Fields of activity at the FCCD: Cell analysis and cell sorting

Cell analysis

Using a cell analyzer, cells can be analyzed electronically due to their biological and physical features. These features can be measured using scattered light and an antibody-fluorescence-dye. The antibody is coupled to fluorescent dye that fluoresces when excited with monochromatic laser light.

The high sensitivity of the cell analyzer allows the detection of as few as 50 molecules per cell. Various cell characteristics are measurable due to the attachment of the antibody to the cell. This type of procedure is also possible for fluorescing proteins (e.g. GFP, RFP) located inside a cell.

A cell analyzer combines three systems: fluidics, optics and electronics. In the fluidics system cells are carried through the flow cytometer and through a cuvette passing a laser beam. The result is a fluorescence signal, which is dispersed in the optics system into its spectral components. Highly sensitive detectors (PMT's) measure the components and generate electrical impulses. These impulses are digitalized and evaluated by a computer.

The FCCF operates three cell analyzers: two LSRII and a FACS-Calibur. The LSRII allows the measurement of up to 15 parameters of single cells. The FACS-Calibur measures up to 6 parameters and is equipped with a special loader; it allows samples to be taken directly from 96 well plates.

Cell Sorting

Cell sorting is another technique used in flow cytometry, whereby cells of interest can be sorted out and collected for further biochemical purposes or functional analyses. Due to their features, single cells can be sorted out selectively from a heterogeneous mixture. At first, these cells are analyzed (as in the cell analyzer) and packed into drops. The sorting of the cells is than accomplished by a specific electrical charge. The charged drop is then deflected by charged electrodes into waiting sample tubes.
The cell laboratory operates four different cell sorters: two FACS-Arias, a FACS Diva, and a MoFlo. The FACS Aria is the most modern and sensitive instrument. It can measure up to 11 parameters and is equipped with digital electronics. The FACS Diva also comprises of digital electronics and is able to measure up to 10 parameters.

The cell sorter MoFlo can measure up to 11 parameters and is equipped with analogue electronics. All cell sorters are high-speed sorters (~20.000 events/s) and allow the simultaneous sorting of four different cell populations.

Perspectives

The number of colors of fluorescence used in flow cytometry for distinguishing small cell-subpopulations increases steadily. This results in the necessity of optimizing experimental techniques and instruments used in Flow Cytometry.

Cellevator

In cooperation with the company OLYMPUS LIFE SCIENCE RESEARCH EUROPA GMBH we developed the Cellevator and were able to implement it into the cell sorter BD FACS Aria. This device effectively prevents cell settlement during the cell sorting process.

EMBO-Practical Course on Cytometry and Cell Sorting for Functional Genomics and Proteomics

Together with various groups working at the DRFZ we offer workshops for users from around the world. In 2008, the EMBO-Practical Course on Cytometry and Cell Sorting for Functional Genomics and Proteomics took place once again, as already in the years 2006 and 2003.

Long Night of the Sciences in Berlin

Every year the cell laboratory is opened to the public under special safety measures. Visitors are offered an insight into highly technical science performed at the DRFZ.

References

Kaiser T, Raba K, Scheffold A, Radbruch A. A sheath-cooling system to stabilize side-streams and drop-delay during long term sorts for FACS Aria cell-sorter.

Kaiser T, Raba K, Sickert M, Radbruch A, Scheffold A. Integration of an ultrasonic wave device in a FACS-Aria cell sorter for continuous, non-invasive mixing of cell suspensions.

Acknowlegdements

We would like to express our special thanks to Katharina Raba, for her professional contributions to our Institute, all her efforts and her readiness to help at all times. Katharina Raba left us in September 2008 after eight years of professional assistance.
She moved on to work at the Institute for Transfusion Medicine in Düsseldorf, where she is currently in charge of establishing a Flow Cytometry Core Facility.

Basic course on flow cytometry

Every month we offer a two hour basic course on flow cytometry, which provides training and education for the proper use of the techniques used in flow cytometry. The course is open for all who are interested. Because of the high demand, persons interested should register in the discussion platform.

Sorterclub

The Sorterclub takes place every two weeks and serves as a forum for discussing and planning experimental research with reference to flow cytometry or cell sorting.