Guidelines and Protocols

Guidelines for working in the Flow Cytometry Unit, user access policy.

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

The Flow Cytometry Unit provides flow cytometry analysis and cell sorting services as well as user training for new users of the instruments. Access to the flow cytometers and their reservation calenders for individual users is given after a training session. Training for new users is arranged regularly.

New users should contact the corresponding unit's personnel and discuss their experimental setup before their first reservation. When making a contact, please provide the following information: Full name, e-mail address and phone number, Department or research group, Principal investigator or supervisor (name and address), Billing information (profit center and WBS codes).

New independent users should be acquainted to basic flow cytometry before the user training. We provide all users with practical guidance for working in the facility. Appropriate training for proper use of the instruments is obligatory for all new users. Contact the corresponding unit´s personnel for further information and reservations for training.

Reservation system is available in iLab. Centralized reservation system for all Flow Cytometry Unit instruments for independent users as well as service users is found in 
User guidelines

The guidelines might vary between the facilities. Please contact the corresponding personnel and/or web pages for more information.

Reservation: Remember to do reservations prior to instrument usage and possible cancellations as early as possible. Uncancelled reservations will be charged (see user fees).

Instrument: Short notice or outside office hours you might need to start the instrument yourself; the user should ensure that the instrument is ready to use with sufficient liquids and waste. Take care that all necessary start up and shut down procedures are done according to instrument guidelines if needed. If you make any special changes to the instrument settings, change filters etc., please return the setup to the starting state. Remember to fill the logbook.

In the lab: In case of problems or need of help, contact the personnel. Please, inform the personnel if you notice any of the solutions or disposables running low in the lab. Remember to take copies of all your data (including the fcs files). Virus scanning must be done every time before you may attach your external memory to any computer.

Laboratory Safety:  Normal laboratory safety procedures should be followed. No eating or drinking in the laboratory. Clean the work area after use.

Flow Cytometer safety: Instruments have high power lasers and on sorters the plates are charged while sorting. Avoid aerosols by keeping covers closed all time while running samples. You can also use AMO on most of the sorters if needed. More information from personnel.

Samples: Contact personnel for more precise instructions. The general rules are:

Samples above BSL-1 are not allowed:

  • 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) has to be certified RCV negative (you can visit ) or fixed for analysis
  • Bacteria: discuss with personnel.
  • iPS and other stem cells: discuss with personnel.
Introduction to Flow Cytometry

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

Modern Concepts of Flow Cytometry Barbara Roth (editor)
Introduction to Spectral Flow Cytometry

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

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 facilities and expertise of Flow Cytometry unit, supported by the Faculty of Medicine of University of Helsinki, HiLIFE and Biocenter Finland, is gratefully acknowledged.” 

e.g. for Viikki: “The facilities and expertise of Flow Cytometry unit, supported by the Institute of Biotechnology of University of Helsinki, HiLIFE and Biocenter Finland, is gratefully acknowledged.” 

Kindly inform the personnel about your publication.

 

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

 
Protocols

: The Power of Reagent Titration in Flow Cytometry

(Paper on Cytometry Part A Journal)

 
Spectral tools

 
Panel design

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

 
Sample preparation

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.