Flow Cytometry (FC) protocol

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This is a general protocol, serving as a guide to the different steps of flow cytometry. Optimise times, volumes, concentrations and reagents for your specific experiment.


  • Harvest the cells and wash them in a single cell suspension.
  • Adjust he cell number to a desired concentration using ice cold FACS buffer.
  • If a functional assay is performed, skip the sodium azide as it can interfere with cell function.
  • Ensure cell viability is around 95%.
  • Stain the cells on ice with cold reagents, so the cell membrane stays in place and proper staining occurs.
  • If the observed cells express high number of Fc receptors, introduce a blocking antibody (e.g., FC-block CD16/32).
  • Incubate on ice for 20 mins.
  • Introduce the primary antibody and incubate the samples. Optimise time and temperature for the specific experiment.
  • Wash cells 3 times, centrifuge them for 5 mins at 1500 rpm and resuspend in cold FACS buffer.
  • If using a secondary antibody, dilute it in FACS buffer according to the supplier’s instructions and resuspend the cells in the solution.
  • Incubate for 30 mins in the dark, followed by a centrifuge and wash/resuspend in FACS.
  • Analyse cells as soon as possible.