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 Fees and Reservations.
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 out side 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.

Flow Cytometer safety: Michael G Ormerod: Flow Cytometry - A Basic Introduction - Appendix 4 Safety

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 samples from screened persons only
  • No viruses,  all cell lines transducted with virus (e.g. lentiviral vectors also 3rd generation) has to be certified RCV negative. (you can visit BVC)
  • Bacteria: discuss with personnel.
  • iPS: discuss with personnel.
Introduction to Flow Cytometry

Thermo Fisher Introduction to Flow Cytometry tutorial

Bio-rad Introduction to Flow Cytometry - Basics Guide

BD Introduction to Flow Cytometry

OpenFlow Cytometry (youtube channel)

Cytometry Part A -Journal

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

BD biosciences Spectral Flow Cytometry

Thermofisher Scientific Spectral Flow Cytometry Fundamentals
Publishing Flow Cytometry data

Please note that we expect you to acknowledge the Flow Cytometry Unit when publishing results using any of the instruments or services. e.g. “The flow cytometry analysis was performed at the HiLife Flow Cytometry Unit, University of Helsinki.” Kindly inform the personnel about your publication.

 

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

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773297/

https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/0471142956.cy…

 
Protocols

 

Single cell suspension from Solid Tissue (Cytometry Part A Journal)
Spectral tools

ThermoFisher Fluorescence SpectraViewer

BD biosciences Fluorecence Spectrum Viewer

Biolegend Fluorescence Spectra Analyser

 
Panel design

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

ThermoFisher Flow Cytometry Panel Design

BD Biosciences Panel Desing

Biolegend Multicolour staining guide

 
FAQ for 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…

BD biosciences web page Panel design  

  • 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. Biorad Permeabilization and Fixation

Thermofisher Cell Preparation for Flow Cytometry Protocols

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.