Cell Sorting Guidelines
High-speed cell sorting can produce aerosols that may present a health hazard to facility members and users. The guidelines are designed to protect the users and staff of the FACS Core Facility (FCF), ensure the proper use and maintenance of the equipment and to avoid cross-contamination.
The FACS Core Facility (FCF) is located in the 10th floor in the Biozentrum. Sorters are situated in the room 10.040. The room is approved for Biosafety Level 2 (BSL-2) work. Correct PPE must be worn and proper containment procedures must always be used in this location.
Due to the complex nature of cell sorting, sorts are performed by designated operators on the AriaIII and Aria Fusion cell sorters. Unassisted use of the Melody cell sorter is possible after proper training. Orientation and additional training is provided by the facility staff. The users need to be familiar with the registration system and the guidelines.
Booking and Billing
The facility is available for assisted sorting on Monday-Friday 10am-6:00pm. The instruments can be booked 21 days in advance. The instruments can be maximally booked for six hours per day. If a user needs more time on an instrument or needs to plan the booking more time in advance, this can be arranged after discussion with the staff.
Fees will apply for the assisted and unassisted sorting, which are calculated based on the reserved time on the online calendar:
- Inhouse members:
- Assisted Mon-Fri (10am-6pm): AriaIII/Melody CHF 30/h; AriaFusion CHF 40/h
- Unassisted: Melody CHF 20/h Mon-Fri (10am-6pm); Melody CHF 15/h after office hours
- Melody Training: CHF 60/h
- Unibas members:
- Assisted Mon-Fri (10am-6pm): AriaIII/Melody CHF 40/h; AriaFusion CHF 50/h
- Unassisted: Melody CHF 30/h Mon-Fri (10am-6pm); Melody CHF 20/h after office hours
- Melody Training: CHF 60/h
- Companies: assisted sorting and prices upon request and availability
Sorting will be charged per 30 minutes. The invoices are sent annually in the second annual quarter. Charging starts upon the start of the scheduled appointment, not when the user enters the lab and it ends when the sort ends. Users must cancel their reservations 12 hours in advance of their scheduled appointment to avoid being charged for the entire time scheduled. If you must cancel your reservation within 12 hours due to unexpected experimental or health issues, you may inform the FCF staff and ask to have the reservation removed and the fee waived. The core reserves the right to restrict your access to the facility in the event of frequent last minute cancellations, late arrivals, no-shows or any other severe disregard of the guidelines.
Approval to sort
New users of the facility need to register for an account on our booking homepage PPMS and fill out and submit the “Cell Sorting Approval Form” that can be found on the PPMS booking homepage prior the first sort appointment. This form needs to be resubmitted and confirmed any times a new experiment is initiated or the experimental protocols are altered a lot. Before the first sort it is highly recommended to contact the FCF and discuss the details of the experiment with the facility staff. Important issues are sample preparation (e.g. single cell suspensions, cell washing, cell concentration), sample transportation (e.g. RT, 4°C, darkness) and cell size, type and sensitivity (e.g. nozzle tip considerations, sort time, sort buffer, antibiotics). Sort requests can be booked using the online booking schedule PPMS. Each entry must include user name, sample description, sample number and for BSL-2 sorts a clear BSL-2 notification. The facility staff has the right to deny sort requests, if conditions have not been clearly disclosed by the user. It is recommended to test new experiments upon fluorescence positivity on a flow cytometry analyzer or by fluorescence microscopy before a sort is scheduled.
Radioactively labeled as also viable samples beyond BSL-2 when in aerosol, cannot be brought to and processed at the facility. Primary human cells as also human cell lines need to be screened and characterized to be free of blood borne pathogens including HIV, Hepatitis B and C viruses and pathogens used to transform cells such as EBV and human papilloma viruses. In this condition they can be sorted using BSL2 with enhanced precautions. Otherwise they are considered to be potentially infectious and aren’t approved for sorting. Also of particular concern are samples containing recombinant DNA, especially if they harbor modifications that result in expression of a toxin or known oncogene or allow the virus to integrate into host genome. There are various modifications to the HIV-based vector and packaging systems in order to improve biosafety. Hence third generation, self-inactivating vectors are considered to confer a higher level of biosafety and therefore are recommended for biosafety level BSL2 whereas first and second generation vectors would demand at least sorting at BSL2 with enhanced precautions.
Only approved sort samples can be delivered to the facility for sorting. Sample transportation must strictly follow the biosafety standards. The cells should be brought in 15 ml tubes or 12x75 mm standard FACS tubes (preferably polypropylene tubes if the cells attach easily). Samples must be completely processed, stained and ready-to-acquire prior sort procedure. Staining steps and filtration, which do not need washing steps (i.e. live/dead stain) can be completed in the facility. It is recommended that the cells are counted before they are brought for sorting. This will allow an estimation of efficiency and the possibility to trace problems with low yield. Samples need to be filtered through a mesh cell strainer smaller than 50um or a cell strainer cap which is suitable for standard FACS tubes, before placing them on the sorter. This can be done in the facility for BSL-1 sorts. BSL-2 samples need to be filtered at the user’s site and must be delivered in a screw-cap tube. The users are responsible to bring the appropriate collection devices with some medium or buffer. Data generated during sorting will be made available to the user within 24 hours after collection.
Please acknowledge the FCF in the appropriate way in publications and grant applications where equipment and/or services and expertise provided by the FCF staff contributed to the final work. Please add newly accepted papers in your PPMS account under ‘Publications'.