Flow Cytometry Unit (FCU) Infrastructure for high-end FACS analysis and state-of-the-art cell sorting capabilities. Facility
The FCU offers access to high-end FACS analysis and state-of-the-art cell sorting capabilities, actively providing scientists from abroad and the UMCG with adaptive setups and dedicated support. The facility also provides services that include training, technical support and assistance with experimental design and data analysis.

Booking information

Before you can use any instrument, you have to:

  • Register as a facility user
  • Follow an individual training
  • Create a personal account in Booked scheduler
  • Read all FCU rules and fee calculations.
    • Complete and submit the online registration form
    • Make sure to upload a digital proof of the ML-II e-learning (pdf, jpg, png or bmp) showing your name and the score.
    • After registration, new users will receive an email for a training appointment by one of the operators on the requested flowcytometer.
    • For the MoFlo Astrios and MoFlo XDP an introduction is not necessary, as these systems are always controlled by the facility operators.
  • The facility uses a booking system for the reservations of the different flowcytometers. All users need to complete an account.

    Once the introduction has been completed successfully, you will get access to the flowcytometer on the booked scheduler agenda.

  • The following rules and regulations apply in general to the people working at the FCU (ML-II classified):

    • Access to the FCU is restricted to those who are aware of the risks involved in entering this facility.
    • Work must not commence until the ML-II e-learning module has been completed and instruction has been given by the Responsible Staff Member (RSM) or another member of staff authorized by the BSO (e.g. the SM).
    • Maintenance staff must consult the RSM or SM before entering the room. All persons working with GMOs at level II must be registered with, and have the approval of, the BSO. No exceptions are made for students or guest staff.
    • All persons working in an ML-II area must comply with additional rules, measures or advice given by the BSO, RSM or SM.
    • In all cases not covered by these rules and regulations, the RSM, SM or BSO should be consulted.
    • Every lab worker is required to inform the RSM, SM or BSO in the event of irregularities, incidents or emergencies.
    • In the case of experiments with radio-nuclides, permission is also required from the Radiation Protection Unit.
    • If activities involving non-GMOs or GMOs classified under ML-I are carried out in the ML-II room, the ML-II work instructions must be applied to all activities.
    • If work has to be carried out in the ML-II laboratory involving microorganisms in risk Class 3 (ML-III), permission must be sought from the BSO.
    • Work involving GMOs must be specified in the GMO permit. The GMO permit contains additional instructions that must be observed.
    • In certain cases, persons who are suffering from a digestive disorder, have an open wound or are taking medication that lowers the immunity will not be permitted to carry out work (consult the BSO).
  • Your time slots in the booked scheduler will be used for fee calculations. The invoice will be send to the responsible Principal Investigator (PI) at the end of the year.

    The invoice for internal users will be calculated as follows:

    • FacsVerse; Quanteon: 5 €/hr
    • LSR-II; FacsSymphony A50; Cytek Aurora: 10 €/hr
    • MoFlo XDP; MoFlo Astrios: 30 €/hr Sony
    • SH800s: 20 €/hr

    Users from outside the UMCG/RuG :

    • Analyzers: 200 €/hr
    • Sorters: 300 €/hr

A-analyzers

    • The Quanteon is a 4 laser flow cytometer that is configured with 25 independent silicium based detectors (SiPM) for meeting demanding sample panels. The SiPM detectors provides excellent sensitivity, stability and a 7.2 log dynamic range! Very dim signals and very bright signals can all be captured and gated in the same view, therefore eliminating the need for laborious trial and error PMT tuning procedures. Small vesicles like bacteria, platelets or even extracellular vesicles can be detected as small as 0,1 um (size based on polysterene beads).
    • Excellent fluidics provide steady and consistent sample delivery, with high reproducibility and exceptionally low CVs even on high flow rates! As the Quanteon has volumetric fluidics absolute counts, without the use of expensive beads, will save time and money.
    • The Novosampler enables easy automated measurement with a tube rack (40 tubes) or various types of 96 wells plates (no deep well) or even a 384 wells plate. Additional cooling racks are available for the tube rack and plate loader.
    • The CFU has 2 identical Quanteons which can serve as their backup in case of technical problems. Please be aware of small differences in sensitivity and gain settings between the two systems!
    • Lasers (OBIS)
      • Violet: 404nm, 100mW
      • Blue: 488nm, 100mW
      • Yellow-green: 561 nm, 100mW
      • Red: 640nm, 100 mW
    • The BD FACSVerse cytometer is a compact research flow cytometer with three lasers (405, 488 and 640 nm) that provide the ability to analyze up to 10 parameters (FSC, SSC and 8 colors ( 4 on the 488 laser, 2 on the 640 laser and 2 on the 405 laser ). Emission filters can be changed and filters are automatic detected. Also pulse processing (height, width and area) can be measured on all the parameters. The vacuum driven fluidics can measure at high (120 µl/min), medium (60 µl/min), and low (12 µl/min) flow rates.
    • Besides manual delivering the sample to the BD FACSVerse cytometer for acquisition, multisampling of samples using the BD FACS Universal Loader can be performed by using 30- and 40- tube racks (5 ml tubes) or multiple types of microtiter plates (96 wells plates)
    • Samples are acquired ( 18 bits digitalization in 5 decades) using the FACSuite software. Samples are saved as FCS3.0 files by which compensation can even be changed after measurement.
    • By using the BD FACSuite CS&T research beads, cytometer performance is checked. Automatic adjustments, ensuring consistent values from day to day and experiment to experiment is possible by calibrating with the CS&T beads (Reference or tube settings can be updated with CST beads to correct for instrumental variations)
    • For more specifications on lasers and detectors, please contact us (find contact details below).
    • The BD LSR-II cytometer is a research flow cytometer with five lasers that provides the ability to analyze up to 20 parameters (FSC, SSC and 18 colors). On these 5 lasers we can measure different fluorochromes (2 on Blue, 3 on Red, 6 on Violet, 4 on Yellow-Green and 3 on the UV-laser). Bandpass and Dichroic filters can be changed with the help of one of the operators (This can only be done by the user themselves after permission of the facility). Pulse processing (height, width and area) can be measured on all the parameters. The pressure driven fluidics can measure at high (120 ul/min), medium (60 ul/min), and low (12 ul/min) flow rates (fine adjustment is possible).
    • Samples are acquired (18 bits digitalization in 5 decades) using DIVA 8.0 software. Samples can be saved as FCS 3.0 or 3.1 files. Compensation afterwards is always possible.
    • For more specifications on lasers and detectors, please contact us (find contact details below).
    • The BD FACSymphony™ SORP is a conventional cell analyzer. The CFU has a 30 parameter system with in total 8 laser. To improve sensitivity and resolution the Symphony is configured with 8 hot PMT’s in the higher wavelengths.
    • The additional HTS (High Throughput System) enables automation for measuring in 96 wells plates.
    • Pulse processing (height, width and area) can be measured on all the parameters. The pressure driven fluidics can measure at high (120 ul/min), medium (60 ul/min), and low (12 ul/min) flow rates (fine adjustment is possible).
    • Samples are acquired (18 bits digitalization in 5 decades) using DIVA 9.0 software. Samples can be saved as FCS 3.0 or 3.1 files. Compensation afterwards is always possible.
    • For more specifications on lasers and detectors, please contact us (find contact details below).
    • The Aurora is a spectral analyzer that makes it possible to detect at least 35 different colors on the same celltype. A 40 parameter parameter panel recently be established (OMIP 69- ref)
    • As in a conventional flowcytometer every fluorochrome is dedicated to just one channel the Aurora uses all 64 channels. No need for changing filters anymore and a very high flexibility for choosing labels. As the whole spectrum is analyzed unusual combinations like GFP and FITC (and many others) can be extracted from each other.
    • The system is equipped with 5 flat top lasers and has 64 channels for detection of fluorescence. Electronics and the APD detectors are very sensitive (low noise) and linear over a wide range (7 log decade)
    • A unique property is the ability to extract the autofluorescense from cells making it possible to increase resolution for high autofluorescent cells.
    • Small particles detection limit is about 110 nm on polysterene beads (same index as EV’s)and 180 nm on silica beads.
    • Lasers:
      • UV: 350 nm, 20 mW
      • Violet: 405 nm, 100 mW
      • Blue: 488 nm, 50 mW
      • Yellow-Green: 561 nm, 50 mW
      • Red: 640 nm, 80 mW

B-sorters

    • The MoFlo XDP is a high speed sorter with 4 lasers (488 nm, 532 nm, 404 nm and 642 nm Lasers. It provides the ability to analyze/sort up to 13 parameters (FSC, SSC and 11 colors). According to the technical specification it can achieve a validated sort rates of up to 70.000 events per second . In our practice, sorting rates till 25.000 events per second are achieved with good recovery, yield and viability using the standard 70 nozzle. Higher speeds (30.000-35.000 events/sec) are very well possible but will lead to lower yield (because of Poisson statistics). 4 different populations can be sorted at the same. Each population can be sorted in its own sort mode (Single, Purify and Enrich).
    • Optional nozzles sizes (100 um, 120 um and 150 um) can be used besides the standard 70 nozzle, to sort bigger cells (this prevents clogging of the nozzle and creates better side streams). On the backside it will reduce the speed a lot (max 8000 cells/sec with the 100 um nozzle), but because of lower shear stress involved it is more gentle for the cells.
    • Multiple tube formats can be applied for sorting using the temperature controlled SmartSampler. The sorted cells can be collected in multiple tube formats like eppendorf tubes and 5 ml tubes. The Cyclone enables sorting in different plateformats (6, 12, 24, 48,60 (Terasaki) 96 and 384 wells) and also on slides. Samples are acquired (32 bits – 5 decades) using the Summit software. Samples are saved FCS3.0 files. Compensation afterwards is possible.
    • For more specifications on lasers and filters, please contact us (find contact details below).
    • The MoFlo Astrios is a high speed sorter equipped with 7 lasers (355 nm, 405 nm, 488 nm, 532 nm, 561 nm, 592 nm and 640 nm The MoFlo Astrios provides the ability to analyze or sort up to 32 parameters (FSC, 6 SSC and 25 colors). According to the technical specification it can achieve a validated sort rate of up to 70.000 events per second . In our practice, sorting rates till 25.000 events per second can be achieved with good recovery, yield and viability using the standard 70 nozzle. Higher speeds (45.000 cells/sec) are very well possible but will lead to a lower yield (because of Poisson statistics). Six different populations can be sorted at the same time. Each population can be sorted in its own sort mode (Single, Purify and Enrich)
    • Optional nozzles sizes (100 um, 120 um and 150 um) can be used besides the standard 70 nozzle, to sort bigger cells (This prevents clogging of the nozzle and creates better side streams). On the backside it will reduce the speed a lot (max 8000 cells/sec with a 100 um nozzle) but because of lower shear stress involved it is more gentle for the cells.
    • Multiple tube formats can be applied for sorting using the temperature controlled SmartSampler. The sorted cells can be collected in multiple tube formats like eppendorf cups, 5, 15 and 50 ml tubes. The Cyclone enables (single cell) sorting in different plates (12, 24, 48, 60 (Terasaki), 96, 384 wells) and also on slides (customable format).
    • Samples are acquired (32 bits digitalization in 5 decades) using the Summit software. Samples are saved as FCS3.0 files. Compensation afterwards is possible.
    • For more specifications on lasers and filters, please contact us (find contact details below).
    • The benchtop SH800S cell sorter permits sorting of a wide range of cell sizes for many applications using the 70-μm, 100-μm, and 130-μm microfluidic sorting chips. This novel, chip-based design is fully integrated with comprehensive fluidics controls and advanced automation for set-up, acquisition, sort and analysis to make sorting less subjective, more precise and easier to perform.
    • System software is intuitive and supports sorting into tubes, 2 ml epcups and 96- and 384-well plates. The SH800S can sort 2 populations at the same time. The software generates FCS 3.0 and FCS 3.1 files that can be exported to third party analysis software.
    • The sorter is equipped with a class A2 Level II biosafety cabinet to provide protection for the operator and the sorted cells. The biosafety cabinet (CAS Biomat2) is custom designed for the SH800S.
    • The optical design offers four colinear excitation lasers (488 nm, 405nm, 561nm and 638nm) and 6 fluorescence channels. This enables the option to measure 6 different fluorochromes but combinations of probes have to be chosen carefully because the lasers are not separated in time so some combinations like APC-Cy7 and PeCy7 are not possible.
    • For more specifications on lasers and detectors, please contact us (find contact details below).

Analysis software

  • For fast tSNE clustering and many others

  • A very complete but bit complex program

  • Most user friendly

  • Old fashioned but still useful for some users

  • The standard in DNA analysis

Contact

Geert Mesander Senior operator central flowcytometry unit