Flow Cytometry (FC) protocol

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.

Procedure:

• 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.