FACS Sorting Core Unit

Aim of the FACS Sorting Core Unit is to provide interested scientists access to latest equipment of flow cytometry. Researchers receive tailored support for their individual scientific projects.
The FACS Sorting Core Unit is located in the Interdiziplinären Klinik für Stammzelltransplantation. Five sophisticated instruments for analysis (FACS CantoII, FACS LSR Fortessa) and sorting (FACS AriaIIIu and FACS AriaFusion) of cells as well as an Image Stream are available. The FACS CantoII and FACS Fortessa can be used indepently after initital training. Researchers have to acknowledge the user guidelines of the facility. The FACS AriaIIIu and the FACS AriaFusion can only be operated by the staff of the unit.
Expert support and advice can be given on request if capacity is available.

Services

  • Cell Sorting with FACS AriaIIIu and FACS AriaFusion
  • Cell Analysis with FACS CantoII and LSR Fortessa
  • Methodological and technical support
  • Support for the establishment of new applications
      • Multicolor-Analysis
      • Surface markers
      • Intracellular markers
      • Fluorescence proteins
      • Functional and Immuno-Assays
      • Apoptosis
      • Cytokine detection
      • Cell cycle analysis
      • Side population
  • Training on FACS CantoII and LSR Fortessa and, forpeople with longterm FACS-experience on FACS AriaIIIu and FACS AriaFusion
  • Support in Bioinformatics

Cell Analysis

  • FACS Canto II

    By FACS Canto II FACS measurements can be carried out independently or by the core facility staff. The device is equipped with the following lasers:

    • 407 nm: violet laser
    • 488nm: Blue Laser
    • 633 nm: Red Laser

  • FACS LSR Fortessa

    The LSR Fortessa expanded as a new flow cytometer FACS Sorting Core Unit. The device allows, in addition to the blue, violet and red laser a yellow-green laser for better detection with the use of PE-dyes and red fluorescent proteins. Up to 17 colorants are used in a sample.
    By FACS LSR Fortessa FACS measurements can be carried out independently or by the core facility staff. The device is equipped with the following lasers:

    • 405 nm: violet laser
    • 488nm: Blue Laser
    • 561 nm: green laser
    • 640 nm: Red Laser

Cell Sorting

  • 1. Cells are harvested by a sheath flow as beads strung on a string (hydrodynamic focusing)

    2. In the laser intersection the analysis of the cells takes place.

    3. The nozzle separates the jet into individual drops and charges the drops to be sorted on.

    4. The charged drops are deflected in an electric field, and fall into the appropriate receptacle.

  • The following tables show how long a sorting operation. These are minimum hours, 20 minutes should be allocated for the setting of the device. The duration varies with the size of the cells.

    Sortdauer smaller cells

    Sortdauer for small cells (15,000 / sec, Mode:. Yield)
    Sortdauer large cells

    Sortdauer for large cells (5000 / sec, Mode:. Yield)
  • Examples for multicolor analysis

  • The following options are possible with the FACS Aria III and FACS Aria Fusion:

    FACS 15ml tube

    1) separated into 2 populations in 15 ml tubes

    FACS tube
    FACS small reaction tube

    2) seperated into up to 4 populations in FACS-tubes or small reaction tubes

    FACS 96-well

    3) filing in microtiter plates (for example, 96-well)

  • FACS AriaIllu

    This Cytometry one or more cell populations from the suspension can be sorted out (Note: not all cells can be sorted well, sometimes the populations are very sensitive). The sorted cells can be cultivated further.
    The cell sorting on FACS AriaIIIu is handled by employees of the Core Unit. The device is equipped with the following lasers:

    • 407 nm: violet laser
    • 488nm: Blue Laser
    • 561 nm: green laser
    • 633 nm: Red Laser

  • Multi color combination
    Lupe zum Vergrößern des Bildes
    Examples of color combinations in a multi-color analysis.

    The device is equipped with the following lasers:355 nm UV laser

    • 405 nm: violet laser
    • 488nm: Blue Laser
    • 561 nm: green laser
    • 640 nm: Red Laser

Further Equipment

  • Technical data:

    Laser:
    - 488 nm blue laser (high-power)
    - 642 nm red laser
    - 785 nm far red laser
    - 375 nm UV laser
    - 405 nm violet laser
    - 561 nm green laser

    Speed:
    - up to 200 events per sec

    Objective
    -20x, 40x und 60x magnicfication

    Sample characteristics:
    - Volume: 20-200yl
    - Utilization efficiency: up to 95%

Registration

The FACS Sorting Core Unit is open to all researchers at the UKE and other interested researchers. Booking is done through the provided Internet booking system. New users call directly to the specified contact person.
There is usage fee per hour:

• FACS Canto II: 17 € per hour
• FACS LSR Fortessa € 22 per hour
• FACS AriaIII 60 € hour
• FACS AriaFusion 60 € hour

Bioinformatics Analyses

  • Analysemöglichkeit 1

    After acquiring your samples at the flow cytometer and manual gating of the cell populations of interest in FlowJo or Diva, you end up with a (huge) Excel table containing all samples and all cell populations. Creating t-SNE plots and heatmaps from this table gives an overview of your data and points out subgroups of samples with similar immunophenotype. We can also include clinical parameters and sample metadata into this kind of analysis.

  • Bioinformatiksche Analyse 2

    After acquisition, proper compensation and removing trash and unwanted cell populations from your FCS files, this analysis creates t-SNE plots directly from your FCS files. The plots will be colored by expression of all markers included in your panel and by density, resulting in a single-cell overview of your data showing all cell populations you might already know plus maybe cell populations you have not been looking at so far. It is possible to create t-SNE plots for visually comparing samples or sample groups.

  • Bioinformatische Analyse 3

    Just like for analysis type (2), data has to be properly compensated and trash and unwanted cell populations have to be removed from your FCS files. This analysis is then capable of dividing the cells into clusters with distinct phenotypes (comparable to what manual gating in FlowJo or Diva is doing) in an unsupervised manner, resulting in a SPADE tree. These trees can further be analyzed and thereby groups of samples can be compared revealing cell clusters being differentially abundant between the groups.

    Data quality is essential for this kind of analysis, therefore we advise you to contact us prior to conducting your experiment so that the experiment and data acquisition can be planned in an appropriate manner.

  • Logo SFB 1192

    Contact person

    Laura Glau

    Tel.: 7410-51979, Room 04.054

    Email: l.glau@uke.de

Contact

Location:

N27 (Campus Forschung)
4. Etage Raum 61.1

Regine Thiele
Dipl.-Ing.
Regine Thiele
  • Core manager
  • FACS Sorting Core Unit
Melanie Lachmann
Melanie Lachmann
  • Deputy Head
  • FACS Sorting Core Unit
Susanne Roscher
Susanne Roscher
  • Chemical-technical assistant
  • FACS Sorting Core Unit
Julia Spötter
  • Biological-technical assistant
  • FACS Sorting Core Unit

Contact Bioinformatics Analyses