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LighthouseFluorescence Technologies Core FacilityZentrum für Translationale ZellforschungBreisacher Strasse 11579106 FreiburgLighthouse User GuidelinesZentrum für Translationale Zellforschung (ZTZ)Medical Center - University of FreiburgThe Lighthouse Fluorescence Technologies Core Facility is a shared resource laboratorylocated in the Zentrum für Translationale Zellforschung (ZTZ). The ZTZ is a joint buildinghousing laboratories from the Center for Chronic Immunodeficiency (CCI) and from theDKTK/Comprehensive Cancer Center Freiburg (CCCF), including the Department ofMedicine I (Hematology, Oncology, and Stem-Cell Transplantation) (Med. I) of the MedicalCenter - University of Freiburg (Universitätsklinikum Freiburg). Although its primary purposeis to support research laboratories from the above institutes, it is accessible to researchlaboratories from all departments and institutes of the Medical Center, as well as from theUniversity of Freiburg. The services of the facility include: Flow Cytometry and Cell Sorting,Confocal Microscopy, Image Cytometry/High Content Screening, and QuantitativePCR/Droplet Digital PCR.Contact Persons:Dr. Marie Follo, Head of FacilityTelephone: 0761 270 77697e-mail: [email protected] Bodinek-Wersing, MTATelephone: 0761 270 77680e-mail: [email protected] Herchenbach, Diplom BiologeTelephone: 0761 270 63770e-mail: [email protected] -freiburg.de/

Medical Center – University of Freiburg, Lighthouse Core Facility User Guidelines04.03.2020Lighthouse - Equipment OverviewFlow Cytometry and Cell SortingHigh Speed Cell Sorters: Beckman Coulter MoFlo Astrios, 14 color (Lasers: 405 nm, 488 nm, 561 nm, 640 nm) Becton Dickinson FACSAria III, 17 color (Lasers: 375/405 nm, 488 nm, 561 nm, 633nm) Becton Dickinson FACSAria Fusion, 16 color (Lasers:405 nm, 488 nm, 561 nm, 640nm)FACS Analyzers: BD LSR Fortessa (5 machines), from 8 to 17 colors and multiple laser wavelengthsavailable, depending on machine used (355 nm, 405nm, 488 nm, 561 nm, 640 nm) Sony SP6800 ZE Spectral Analyzer, 32 channel spectral detector, spectral unmixing,20 colors, (405 nm, 488 nm, 638 nm) BD FACS Canto 8 color (405 nm, 488 nm, 640 nm) Beckman Coulter Gallios 10 color, (405 nm, 488 nm, 640 nm) Beckman Coulter Navios 10 color (405 nm, 488 nm, 640 nm)Microscopy and ImagingConfocal Microscopes: Zeiss LSM 880 AiryScan Confocal Microscope: AiryScanFast detector, spectraldetector, integrated climate chamber, 4 lasers (405 nm, multiline argon, 561 nm, 633nm) Zeiss LSM 710 Confocal Microscope: spectral detector, integrated climate chamber, 4lasers (405 nm, multiline argon, 561 nm, 633 nm) Leica SP2 AOBS Confocal Microscope: 5 lasers (405 nm, multiline argon, 543 nm,594 nm, 633 nm)High Content and Automated Microscopes: Olympus ScanR High Content Screening Station: inverted widefield system,automated microscopy/image cytometry, integrated climate chamber, numerousfluorochromes/filter cubes available Sartorius IncuCyte S3: widefield system, long term live cell imaging, independentimaging/analysis of up to 6 plates simultaneously, 2 color fluorescence and brightfieldStandard Widefield Systems: Zeiss Axio Imager M2m: upright widefield microscope for transmitted light andfluorescence Zeiss Axio Observer: inverted widefield microscope for transmitted light andfluorescence, Apotome, small, on-stage climate chamber Zeiss SteREO Discovery.v8: stereo microscope, transmitted light, fluorescence(GFP/RFP)

Medical Center – University of Freiburg, Lighthouse Core Facility User Guidelines04.03.2020Quantitative PCR (QPCR) and Digital PCR (dPCR) Roche LightCycler 480 (2 machines), Real-Time PCR Cycler, 384-well and 96-wellblocksBio-Rad QX100 Droplet Digital PCR system (droplet generator, droplet reader)Stilla Naica Crystal Digital PCR System (Naica Crystal Geode (x2), Naica CrystalReader)Analysis WorkstationsThere are several dedicated analysis workstations equipped with offline versions of allmanufacturer/machine specific softwares, including the following programs:Flow Cytometry: BC Kaluza BD FACSDiva 8.x BD FCAP Array DeNovo FCS Express FlowJo X Sony Amateras (spectral analyzer) Verity Software ModFit LTMicroscopy: Bitplane Imaris CellProfiler/CellProfiler Analyst ImageJ/Fiji Media Cybernetics AutoQuant X3 / AutoDeblur (deconvolution) Molecular Devices MetaMorph Premier Olympus ScanR Analysis Zeiss Zen Blue/Zen Black offlineNucleic Acid Analysis: Bio-Rad QuantaSoft /QuantaSoft Pro Premier Biosoft BeaconDesigner 8 Roche LightCycler 480 offlineData StorageData from all cell sorters is backed up and archived for 10 years and copies of the data filesare available upon request. Due to space limitations and the high costs involved, the longterm archival of data from the remaining machines is the responsibility of the individualresearchers. USB sticks or portable hard drives are not allowed to be directly connected toany of the confocal microscopes or the ScanR microscope. Data from these machines is tobe saved directly to the attached analysis computer and then moved to the appropriateserver, or saved directly to the ScanR server, respectively.User FeesFor current Lighthouse Core Facility user fees please see Appendix A.

Medical Center – University of Freiburg, Lighthouse Core Facility User Guidelines04.03.2020Facility AccessAccess to services and equipment of the facility can be granted only after a Lighthouse UserAccess Form has been signed and returned, stating which services are required andacknowledging that these user guidelines have been read and will be abided by.In the case of cell sorting, a Lighthouse Biosafety Form must also be submitted before thestart of every new scientific project. It is the responsibility of the user and group leader tosubmit a new form when the project changes (i.e. addition of a new retroviral vector, etc.).This is necessary in order to determine the potential biohazard risks involved in the case ofaerosol formation, and to protect the safety of the sort operators and users of the facility.Appointments for all of the machines are available on a "first-come, first-served" basis. In thecase of overbooking, preference will be given to groups belonging to the institutes of the ZTZ(Med. I, CCI, CCCF) at the discretion of the head of the facility.With the exception of the cell sorters, users are allowed to book machines once they havebeen trained to use them. Training sessions for the machines occur regularly, and spots canbe reserved by telephone or e-mail by contacting the facility. Once a user becomes proficientand able to work independently on a machine, there is also the possibility to use themachines outside of normal operating hours when necessary. Trained users will be able togain access to the required area of the Lighthouse Core Facility through a specialtransponder.It is possible that user access to the Lighthouse Core Facility service may be denied: if the user does not fulfill his or her responsibilities regarding proper use of theequipment or the facility. if the capacity of the equipment or its operators is insufficient to fulfill the requestedusage. if the operators, users or machines could be harmed through carrying out the plannedexperiment.Booking SystemWith the exception of the high speed cell sorters, each of the machines can be bookedthrough the online calendar, which is available on the intranet of the Medical Center –University of Freiburg and can be accessed from the Lighthouse Core Facility website.Usage of the cell sorters requires the availability of an operator. Therefore, appointments forthe cell sorters are visible on a read-only basis on the intranet calendar, and can be made bycontacting the facility via telephone or e-mail ([email protected])."No show" PolicyWhen possible, users should cancel their appointments at least 24 hours in advance of thescheduled start. "No shows" which have not cancelled their appointment or failed to havenotified the Lighthouse Core Facility in some other way may be billed for their appointmentas originally scheduled.

Medical Center – University of Freiburg, Lighthouse Core Facility User Guidelines04.03.2020AcknowledgementsOur user fees cover only a small fraction of our total running costs. Most of our running costsare in fact paid for by our home institutes (Department of Medicine I, Center for ChronicImmunodeficiency, DKTK/Comprehensive Cancer Center Freiburg(CCCF)), while themajority of our machines were financed with the assistance of outside sources such as theDFG, Land Baden-Württemberg, or BMBF. We therefore depend on properacknowledgements in publications and grants. Publications, posters or presentations thatmake use of Lighthouse services or equipment, or data collected in the facility or by its staffshould list this contribution in the respective acknowledgement sections.An example of a general acknowledgement could take a format similar to the following:We thank the Lighthouse Core Facility staff of the Medical Center - University of Freiburg forhelp with their (cell sorting, FACS, confocal microscopy, etc.) resources and their excellentsupport.If you wish to explicitly thank a member of the staff, just name the particular person(s)involved, here alphabetically listed: J. Bodinek-Wersing, M. Follo, D. Herchenbach.Thank you.

Medical Center – University of Freiburg, Lighthouse Core Facility User Guidelines04.03.2020Appendix ALighthouse Core Facility User FeesFees for the use of Lighthouse services/equipment for academic users are as describedbelow. Non-academic groups outside of the Medical Center – University of Freiburg will becharged VAT (MwSt.), in addition to the non-academic user fees. For information about feesfor non-academic users, please contact us directly.Cell SortingGroups of the Medical Center – University of Freiburg, as well as groups from the Universityof Freiburg or other academic institutes50 / hour 25 setup feeFACS AnalysisGroups of the Medical Center – University of Freiburg, as well as groups from the Universityof Freiburg or other academic institutes, minimum booking time 30 min. 3 laser FACS analyzer15 / hour unassisted 4 laser FACS analyzer/spectral analyzer20 / hour unassistedConfocal and High Content Screening MicroscopesGroups of the Medical Center – University of Freiburg, as well as groups from the Universityof Freiburg or other academic institutes, Zeiss LSM 880 AiryScanFast30 / hour unassisted Zeiss LSM 71025 / hour unassisted Leica SP2, Olympus ScanR20 / hour unassistedOther Microscopes (widefield; long-term live cell imaging)Groups of the Medical Center – University of Freiburg, as well as groups from the Universityof Freiburg or other academic institutes, Sartorius IncuCycte 2 / plate / hour Zeiss Axio Imager / Axio Observer10 / hourQuantitative PCRGroups of the Medical Center – University of Freiburg, as well as groups from the Universityof Freiburg or other academic institutes Roche LC480(user supplies consumables/reagents)10 / standard run (1.5 hr)Droplet Digital PCRGroups of the Medical Center – University of Freiburg, as well as groups from the Universityof Freiburg or other academic institutes, Bio-Rad QX100(incl. Lighthouse consumables/reagents)5 / well Stilla Naica(consumables/reagents not included)5 / Geode, 10 / 2 GeodesFor machines billed on an hourly basis, fees are rounded up to the nearest quarter-hour.Billing is sent out quarterly to the corresponding group leaders.

Sony SP6800 ZE Spectral Analyzer, 32 channel spectral detector, spectral unmixing, 20 colors, (405 nm, 488 nm, 638 nm) BD FACS Canto 8 color (405 nm, 488 nm, 640 nm) Beckman Coulter Gallios 10 color, (405 nm, 488 nm, 640 nm) Beckman Coulter Navios 10 color (405 nm, 488