Guidelines and Protocols

Also basic flow cytometry and find some help for planning your experiments.

Reservation

• Make your plans and reservations well in advance: the instrument is available for the time of your reservation only.  
• If you know you need assistance, also contact the staff as early as possible.
• Inform the staff immediately of any delays.
• If you need to cancel, do so as soon as possible and latest 24h before the reserved time.
• Uncancelled reservations will be charged.
• For short notice reservations and usage outside office hours, be prepared to start up the instrument yourself.
• The last user of the day is responsible for shutting down the reserved instrument.
• Leaving an instrument on overnight will result in charges for the time required to recover the instrument and any resulting downtime.
• Fill the user logbook found by the instrument.

Laboratory safety and correct laboratory behavior

• Normal laboratory safety procedures should be followed. Use gloves and lab coats. No eating or drinking in the laboratory.
• Samples above BSL-1 are not allowed. If you have any doubts, please consult with the personnel. The general rules are:
  • No infectious, pathogenic or toxic samples are allowed.
  • Human blood and primary samples from screened persons only.
  • No viruses; all cell lines transduced with virus (e.g. lentiviral vectors also 3rd generation) must be certified RCV negative (you can visit ) or fixed for analysis.
  • Bacteria: discuss with personnel.
  • iPS and other stem cells: discuss with personnel.
• Instruments are equipped with high-power lasers. Do not look directly into the laser beams.  
• Sorter deflection plates are charged with high voltage while sorting. Do not touch the plates when the red light is on or the voltage ON sign is displayed.
• To avoid aerosols keep covers closed at all times while running samples. Most sorters have an aerosol management option that can be used if needed.
• Always return the instrument to its default state after use (e.g. change of filters).
• Clean the instrument and the work area after use.
• Discard unused samples into the appropriate waste containers. The unit is not responsible for the storage of samples left behind.
• Take copies of all your data (including .fcs files). The unit is not responsible for storing or preserving your data. Files older than one month will be routinely deleted.

Collaboration

When the LSRI head or personnel provides substantial intellectual input to the project, they should be included as authors according to established of the Finnish national board on research integrity. Basic services are available with the same prices independently of the collaboration. Collaboration is agreed between the PI and the head of the Flow cytometry unit.

Acknowledgments

The use of the research infrastructure should be mentioned in the acknowledgements section of publications, for example with the sentence: 

“Flow cytometry analysis/sorting was performed at the Biomedicum/Viikki Flow Cytometry unit, University of Helsinki, supported by HiLIFE and Biocenter Finland.”

Kindly inform the personnel about your publication.

Keep in mind MIFlowCyt:The minimum information about a flow cytometry experiment

Any damage caused to laboratory equipment due to negligent behaviour may be charged. 

Inability to follow these guidelines may result in being banned from using the unit. 

Flow Cytometry Protocols Editors: Hawley, Teresa S., Hawley, Robert G. (Eds.)

Modern Concepts of Flow Cytometry Barbara Roth (editor)

Although workflow is pretty much similar to conventional Flow Cytometry is good to get acquainted with how to prepare for spectral Flow Cytometry

Please note that we expect you to acknowledge the HiLIFE LSRIs Flow Cytometry Unit when publishing results using any of the instruments or services. 

e.g. for Biomedicum: “The flow cytometry analysis/sorting was performed at the University of Helsinki Flow Cytometry Unit, supported by the Faculty of Medicine, HiLIFE and Biocenter Finland.” 

e.g. for Viikki: “The flow cytometry analysis/sorting was performed at the University of Helsinki Flow Cytometry Unit, supported by the Institute of Biotechnology, HiLIFE and Biocenter Finland.” 

Kindly inform the personnel about your publication.

Keep in mind MIFlowCyt:The minimum information about a flow cytometry experiment

: The Power of Reagent Titration in Flow Cytometry

(Paper on Cytometry Part A Journal)

: See the configuration of our instruments and design your panels based on them.

Sample preparation

• Only BSL1 cells; No infectious or pathogenic samples are allowed. All human samples from screened persons only. Transduced cells must be certified RCV negative. Bacterial and iPS samples must be discussed with personnel.
• To remove debris and excess antibody, wash samples in PBS or FACS buffer.
• Cell concentration depends quite a lot on your cells and experiment. With high efficiency and resolution experiments (e.g. Cell cycle, plate sorting and big or “sticky” cells) slower is better, 1 million/ml is usually a quite good concentration. With tube sorting for instance you can test 5-10 million/ml and have buffer on hand to dilute if needed.
• Remember that there should be around 100ul dead volume in the sample (amount in the end that you should not run to prevent air going into the system)
• If you use FACS buffer or PBS with low serum if any (max 2 % filtered). If necessary you can use medium but remember that high amount of serum and phenol red will give some fluorescence background.
• With tissue culture you might need to optimize all the steps based on the tissue in question. Tissue samples tend to give lot of fluorescence background from cell debris. Consider using viability dye.

Analysis

• For set up, it is always advisable to have an unstained control sample and multicolor experiments will need all separately stained controls for compensation.
• Multicolour experiments should be planned thoroughly to avoid unnecessary overspills and compensation spread; Small populations with brighter colours and subpopulations should not have spilling from previous population…

• Remember to use Fc-blockers if needed/provided.
• Always prefer primary antibodies if possible.
• When fixing the samples please keep in mind the experiment in hand; What fixative do you need, do you need to study viability, staining first or after (intracellular or out, some antibodies might lose fluorescence), cells become buoyant so be careful with handling.

Sorting

• Always filter samples before sorting and remember to have tubes and buffer for the sort collection. You can pre-coat the inner walls of the collection tubes with a protein containing solution.
• For collecting few events-1000 cells, use plate. For thousands, up to few hundred thousand you can use 1-2ml tubes. About million cells into 5ml tube and several millions into 15ml tube.
• Consider using viability dye
• Also consider sample temperature; if the cells are standing for long periods of time cooling the sample and instrument (sorters) to 4°C might be a good idea. Plate sorting or fast live analysis might be enough to have in RT.