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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.
|Methanol Sensitive||Methanol Resistant|
|Alexa Fluor 647|
|Alexa Fluor 488|
|All tandem dyes|
Keywords: FACS staining protocol, intracellular staining procedure, perm and fix protocol, flow cytometry antibody staining procedure, FACS antibody, flow validatedClick for more optimization tips