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Experimental Design and Execution

Tips for a successful flow cytometry experiment include:

i)   Know the antigen expression of your cells.

ii)  Know the instrument configuration/filters

     - Check with facility staff the lasers and filters available on each machine

iii) Match fluorochromes according to antigen density

     - Match weaker antigens with a brighter fluorophore and stronger antigens with a dimmer fluorophore

     - Check the stain index of different fluorochromes.

iv) Minimise spillover by spreading fluorochromes over the spectrum/lasers

     - Fluorescence spillover can be estimated by using spectrum viewers such as those from BD, BioLegend or Thermo-Fisher.

     - Avoid choosing fluorochromes with high spillover for markers co-expressed on the same cell

 v) Provide proper compensation controls  

     - Single stained cells 

     - Single stained compensation beads 

vi) Provide proper gating controls

     - Fluorescence-minus-one controls are essential for proper gating in multi-color experiments.   

Users are welcome to schedule an appointment with core staff to discuss your experimental design or requirements. It is good practice to check with core staff regarding availability of filters for fluorochromes that you are intending to stain your samples with. Fluorescence spectrum viewers can provide estimates of the spillover matrix between different fluorochromes for such purposes.