Flow Cytometry Technical Resources

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Flow Cytometry Intra-cellular Staining Optimization

Permeabilization and Fixation Method Tips

  • For the staining of secreted proteins like cytokines, a protein transport inhibitor such as Monensin or Brefeldin A should be added prior to fixation/permeabilization in order to trap the cytokines inside.
  • For combined surface and intracellular staining, it is advisable to first stain for the surface markers and then fix/permeabilize as the latter can alter some antigen epitopes and affect antibody binding.
  • Fixation/permeabilization reagents alter the scatter properties as well as the auto-fluorescence of cells. Therefore it is recommended to include an unstained control that has been treated with the same reagents.
  • Binding of antibody to surface antigen can stimulate the cells and alter the expression of intracellular signalling proteins. Therefore, surface staining should always be performed after the stimulation.
  • For phosflow staining, the cells should be fixed and permeabilized immediately after the stimulation as phosphorylation is a transitory phase and quickly pass.
  • Choosing the right permeabilizing agent is extremely important – one can choose between detergents like saponin or TritonX and methanol.
    1. Saponin does not alter the surface antigen epitopes so surface staining can be done afterwards.
    2. TritonX and Tween should be avoided as they can lyse cells if incubated for long.
    3. Methanol is compatible with most intracellular antigens and cells treated with methanol can be stored at -20 to -80°C for an extended duration without loss of signal.
    4. Not all fluorochromes however can withstand methanol treatment. The table below shows which commonly used dyes are methanol resistant and methanol sensitive.

Methanol Sensitive

Methanol Resistant



eFlour 450


eFluor 660


Alexa Fluor 647

All tandem dyes

Alexa Fluor 488



Keywords: FACS staining protocol, intracellular staining procedure, perm and fix protocol, flow cytometry antibody staining procedure, FACS antibody, flow validated

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