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 Imaging Flow Cytometer 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
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- Multicolor-Analysis
- Surface markers
- Intracellular markers
- Fluorescence proteins
- Functional and Immuno-Assays
- Apoptosis
- Cytokine detection
- Cell cycle analysis
- Side population
- Multicolor-Analysis
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- Training on FACS CantoII and LSR Fortessa and, forpeople with longterm FACS-experience on FACS AriaIIIu and FACS AriaFusion
- Support in Bioinformatics
Cell Analysis
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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
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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
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FACS Sorting - The principle
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.
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Sorting: duration
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.
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Sorting: multicolor experiments
Examples for multicolor analysis
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Sorting: different sample collections
The following options are possible with the FACS Aria III and FACS Aria Fusion:
1) separated into 2 populations in 15 ml tubes
2) seperated into up to 4 populations in FACS-tubes or small reaction tubes
3) filing in microtiter plates (for example, 96-well)
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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
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FACS AriaFusion
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
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Imaging Flow Cytometer
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 laserSpeed:
- up to 200 events per secObjective
-20x, 40x und 60x magnicficationSample 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
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1. Creation of t-SNE plots and heatmaps from data (Excel table) generated by classical manual analysis in FlowJo or FACSDiva
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.
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2. Creation of t-SNE plots directly from FCS files
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.
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3. Prospective analysis of FCS files using SPADE
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.
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Further information und contact person (Bioinfomatics)
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

- Core manager
- Cytometry and Cell Sorting Core Unit

- Deputy Head
- Cytometry and Cell Sorting Core Unit

- Chemical-technical assistant
- Cytometry and Cell Sorting Core Unit
- Biological-technical assistant
- Cytometry and Cell Sorting Core Unit
Contact Bioinformatics Analyses
