Abstract The rapid progress in flow cytometry means that an increasing number of parameters can be looked at simultaneously, which is highly relevant for the assessment of lymphocyte characteristics, in particular their multifunctional profile. However, procedures using less complex technology need now optimization in order to take into account the new reagents and applications related to polychromatic flow cytometry. Through optimization of an immunomonitoring protocol to assess the functional profile of antigen specific T-cells using 9–10 colour flow cytometry, we tested the efficacy of three distinct standardized permeabilization buffers for the staining of relevant intracellular molecules. We show significant discrepancies in staining sensitivity for cytokine and cytotoxic factor expression from one permeabilization kit to another, which can lead to different data and interpretation. It is important to be aware of this potential bias and to design specific application/experimental procedures in order to obtain optimal results.