Home Research Working Groups Flow Cytometry & Cell Sorting (FCCF)

Flow Cytometry & Cell Sorting (FCCF)

State-of-the-art cell analysis and cell sorting through technical innovation

Introduction
Members
projects
Patents
Technical equipment
Cooperation partners
Selected Publications
Service
Innovations

The Flow Cytometry Core Facility (FCCF)provides a comprehensive range of services and proprietary technological innovations to ensure state-of-the-art analysis and sorting of cells. The Central Laboratory, operated by the DRFZ, was established in 2000 and is equipped with a broad spectrum of machines. It is used by more than 300 scientists and clinicians from the DRFZ, the Charité, the Max Planck Institute for Infection Biology and other research facilities.

Flow cytometry is an analytical method used for the quantitative measurement of physical, biochemical/cell biological and immunogenetic parameters of single cells by optoelectronical means. On the basis of these parameters various cell features, such as viability, quantification of antigens, phenotype, function, and cell cycle can be determined.

In a flow cytometer, up to 20.000 cells per second can be analyzed for more than 15 parameters simultaneously and quantitatively using light scattering and combinations of antibodies labelled with different fluorescent dyes. The high sensitivity of the cell analyzer allows the detection of as few as 50 molecules per cell. This technology allow us to monitor patients with rheumatic diseases before and during therapy on a cellular level, aiding us not only in the identification of cellular signature of different rheumatic diseases, prediction of therapy response, and monitoring of therapy response, but may also help to understand the cellular processes underlying rheumatic diseases.

By cell sorting cells of interest can be isolated from complex mixtures and collected for further biochemical or functional analyses. For this, cells are measured (as in the cell analyzer) and subsequently packaged into individual droplets. The droplets are then given an electrical charge according to their staining pattern and then deflected by charged electrodes into waiting sample tubes.

In combination with suitable preenrichment methods, such as magnetic cell sorting, also rare cells, such as antigen-specific T cells can be analysed and isolated with high purity for subsequent molecular analyses. For the isolation of sensitive cells, such as plasma cells or stroma cells from the bone marrow, the FCCF utilizes a BD Influx™ sorter with adapted high-speed sorting protocols that ensure high viability and purity of the cells.

To improve the sensitivity of multiparameter flow cytometry we are, in cooperation with the company APE, working on the hardware development for a new multispectral Flow Cytometer. A spectral flow cytometer detects the entire spectra of an individual fluorescence labelled cell, instead of only detecting a specific peak of a certain fluorochrome which has passed through an optical band-pass filters, and thus collects more light increasing the detection sensitivity (Feher et al., 2016).

In cooperation with APE we have also developed a LED based calibration tool (quantiFlash™) which enables us to perform quality control of our machines with respect to the dynamic detection range as well as the signal-to-noise ratio (Giesecke et.al., 2017). It also allows us to calibrate the measured light intensities of a flow cytometer to absolute values. By this we are now for the first time able to quantify absolute numbers of proteins expressed by a cell.

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Members

Group leader
Dr. habil. Hyun-Dong Chang

Technical Manager
Dipl. Ing. Toralf Kaiser

Operators
Dipl. Biochem. Jenny Kirsch
Ana Catalina Teichmüller (M. Sc.)

Scientists/project team members
Dr. Kerstin Heinrich (Biology)
Daniel Kage (M.Sc. Physics)

 

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to projects

EU-gefördertes Drittmittelprojekt „PULZY“
Nahezu 2 Millionen Menschen in Deutschland leiden an entzündlich- rheumatischen Erkrankungen, darunter sind 20.000 Kinder. Um die Ursachen für diese Erkrankungen zu verstehen sind Messmethoden notwendig, die krankhafte Veränderungen an Zellen robust aufspüren und erkennbar machen. Solche Untersuchungen werden bislang häufig mit durchflusszytometrischen Methoden durchgeführt, die aber immer als Voraussetzung eine aufwändige Probenaufbereitung und das Anfärben der zu untersuchenden Zellen mit zellspezifischen Molekülen haben. Die dabei benötigten Prozeduren und Reagenzien können unerwünschte Auswirkungen auf die Zellen haben, wie z.B. Aktivierung, Beeinflussung immunphänotypischer Merkmale, Zellpermeabilisierung, Zellverlust oder Apoptose (Greve et al., 2006; Smiljanovic et al., 2012; Westendorf et al., 2014). Desweiteren weisen derzeit verfügbare Durchflusszytometer Einschränkungen im Hinblick auf die Anzahl messbarer Parameter, Messgenauigkeit, Sensitivität, Vergleichbarkeit und Reproduzierbarkeit sowie Stabilität der Messung auf (Giesecke et al., 2017; Wang et al., 2017)
Im Rahmen des Forschungsprojekts „Pulsformbasierte Durchflusszytometrie und Zellsortierung“ sollen beide Punkte durch eine neuartige Analysemethode der pulsformbasierten Durchflusszytometrie adressiert werden. Ziel dieses Projektes ist die Erforschung, ob anhand einer Pulsformanalyse der Streulichtsignale Zellen identifiziert werden können, ob in Kombination mit der konventionellen Durchflusszytometrie eine Qualitätsverbesserung der Daten erreicht werden kann und unter welchen Voraussetzungen eine markerfreie Zellanalyse und Zellsortierung durchführbar ist. Das Forschungsprojekt wird in Kooperation mit der APE Angewandte Physik und Elektronik GmbH durchgeführt und hat eine Laufzeit von 3 Jahren (Beginn 01.10.2018).

Dieses Forschungsprojekt wird im Rahmen des „Programm zur Förderung von Forschung, Innovationen und Technologien (Pro FIT)” gefördert. Die Zuwendung wird aus Mitteln der Europäischen Union (EFRE) und des Landes Berlin kofinanziert (FKZ: 10165404).

EFRE-Logo

Projektbeteiligte:

Toralf Kaiser
Claudia Giesecke-Thiel (MPI molgen)
Conrad von Volkmann (APE)
Kerstin Heinrich
Daniel Kage
Jenny Kirsch

 

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to
Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Patents
  1. Kaiser, J. Kirsch,
    A. Grützkau,
    Patent number 9958393 :
    ‘Principle component analysis (PCA) – based analysis of discontinuous emission spectra in multi-chromatic flow cytometry’
  2. Kaiser, T. Kolbe, M. Kneissl,
    Patent number: 9498550
    ‘Flow cytometer desinfection module’
  3. Kristen Feher, Toralf Kaiser, Konrad von Volkmann, Sebastian Wolf
    Publication number: 20170322137
    ‘Method and system for characterizing particles using a flow cytometer’

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Technical equipment

1 Analyser MACSQuant (3-lasers) bobby_configuration

1 Analyser MACSQuant (3-lasers) MACS Quant Camilla

1 Analyser LSRFortessa (4-lasers)Wayne 6v2b4y3r 17022016

1 Analyser FACS Canto (3-lasers) Canto New

1 Analyser Symphony A5 (5-lasers) Janice 5b8v3r5yg7uv 23022017 1

1 Analyser Attune NxT (4 Laser)

2 Cell Sorter FACS Aria (3-lasers) ARIA Rowlf

1 Cell Sorter FACS Aria II (4-lasers) AriaII Floyd

1 Cell Sorter Influx (5-lasers)

1 Cell Sorter Sony MA900 Konfiguration Sony MA900

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Cooperation partners

Max Planck Institute for Infection Biology, Berlin

Technische Universität Berlin

Angewandte Physik und Elektronik GmbH (A•P•E), Berlin

MPI for molecular genetics, Berlin

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Selected Publications

Giesecke, K. Feher, K.v. Volkmann, J. Kirsch, A. Radbruch, T. Kaiser, “Determination of background, signal-to-noise and dynamic range of a flow cytometer – a novel practical method for instrument characterization and standardization”, (under review)

Feher, K. von Volkmann, J. Kirsch, A. Radbruch, J. Popien, and T. Kaiser, “Multispectral flow cytometry: The consequences of increased light collection,” Cytometry A, Jun. 2016.

Comparison study of various flow cytometers using a novel ultra stable calibration light source. von Volkmann, K., Feher, K., Popien, J., Kaiser, T. Poster at DGfZ meeting, Berlin, 2015.

Classification of Flow Cytometry Samples with a Dimension Reduction and Binning Approach. Feher, K., Radbruch, A., Kaiser, T. Poster at Cyto conference, Glasgow, UK, 2015

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Service

To create a new account, please fill out the registration form Userform and hand it to a staff member. After registration, the user gets access to the online-scheduler. There, the use of equipment, the documentation of experiments, and billing can be managed:

https://eu.ilabsolutions.com/service_center/show_external/3558

We also provide the central data server “Totoschka” for users in order to store FACS data. The data is accessible online.

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Innovations

quantiFlash(TM)

In cooperation with the Berlin based company A.P.E. we developed quantiFlash. QuantiFlash is a LED based light source for scale calibration. This tool allows quantitative comparison of flow cytometers.

Multispectral Acquisition and Analysis of Flow Cytometric Data Using a Conventional Flow Cytometer

We introduced an experimental strategy, in which discontinuous emission spectra were acquired and analyzed by principal component analysis (PCA). In a proof-of-principle study we could show, that the simultaneous detection of up to 4 fluorochromes, which were all excited by a 488 nm Argon-laser could be resolved by the detection of a discontinuous emission spectrum. In other words we use a standard cytometer just by changing the optical filters as a multispectral cytometer. No further expensive modifications as described in the literature are required. For simplifying data processing, PCA algorithm have to be implemented in standard flow cytometry software. Altogether, our studies demonstrated for the first time new possibilities in combining fluorochromes with similar emission spectra.

A UV-C LED Sheath Fluid Desinfection Module for Flow Cytometric Cell Sorting

We have developed a flow through reactor, which sterilizes the sheath fluid at the point of use. The flow through reactor is comprised of a 35 LED array positioned on top of three concentrix channels through which the sheath fluid passes. We use Galliumnitrid (GaN) based UV-C (220-290 nm) LEDs, which have attracted increasing interest as novel UV-C radiation sources for applications in water purification and desinfection.
Compared to conventional gas-discharge lamps, UV-C LEDs have a compact form factor, operate at very low DC voltages, exhibit fast on/off switching capabilities, and their emission wavelength can be tailored to the optimum wavelength for germicidal effectiveness. We have installed the module in a FACS Diva cell sorter and have connected it to the sheath fluid tubing close to the nozzle. At a sheath pressure of 30 psi, we were able to reduce the number of microorganisms up to 1000-fold.
The cell sorter remained free of microorganisms for at least 7 days after the sterilization with bleach was performed.

Device to cool the sheath fluid on a FACS Aria

We developed a cooling device in order to keep the sheath fluid temperature on a FACS Aria constant. We found that the foccusing of the side-streams on a FACS Aria are temperature sensitve. Room and instrument temperature shifts of more than +/- 5K results in a defocusing of the side-streams and the recovery of sorted cells decreased drastically. Furthermore, the sensitivity of delayed lasers decreased due to instable laser-delay.
To overcome this effects we hold the sheath-fluid constant at 17°C by using our device.

Non invasive mixing of cells during long-term sorts

During long-term sorts cells often sediment at the bottom of the sample tube. In consequence, the threshold rate are instable or interupted and cells in the settlement are exposed to cytotoxin. In cooperation with Advalytix we modified their universal acoustic mixing platform “SAWStation4” so that we can implement it in a FACS Aria.
In our study we could show that the yield of sorted cells was about 5% higher compare to cells mixed by the original FACS Aria agitator. Moreover, we found less dead cells in sorted populations.

 

Group Leader
    Dr. habil. Hyun-Dong Chang
Continue to Introduction