Method for improved detection of antigen-specific immune cells isolated from tissues, comprising the following procedure steps: - isolating of vital immune cells from tissue using appropriate preparation techniques, for example, using enzymatic or mechanical extraction, - determining the total cell count, for example 10 4 to 1.5x10 6 cells are needed per stimulation batch, - prestaining of the isolated immune cells using a fluorochrome-coupled antibody which is aimed at the antigens expressed on the cells, e.g. CD45, or using fluorochromes capable of staining vital cells, e.g. carboxyfluorescein diacetate succinimidyl ester, CFDA-SE, - removal of unbound antibodies or fluorochromes by washing steps, - mixing of the prestained immune cells with heparinized whole blood of the same individual patient, - stimulating the mixed samples with the interesting antigen, with or without addition of co-stimulatory antibodies anti-CD49d and anti-CD28, anti-CD49d alone or anti-CD28 alone, - incubation for 30min to 4h at 35 to 39°C and 3 to 10% CO 2, - adding of a secretion inhibitor, e.g. brefeldin A, monensin or others, - incubation for 2 to 12 hours at 35 to 39°C and 3 to 10% CO 2, - lysis of erythrocytes and fixation of the leucocytes, - washing steps for isolating the leucocytes, - increasing cell membrane permeability by addition of a cell membrane permeability increasing substance, e.g. saponin, - incubation with specific fluorochrome-marked antibodies against activation markers, e.g. anti-CD69, anti-IFN-y, for at least 10min on ice or at room temperature or at 35 to 39°C, - repeated washing steps for removing unbound antibodies, - analysis of cells on a flow cytometry device and discriminating the immune cells from tissue against those from blood based on the fluorescence signal which was used for the prestaining, e.g. fluorochrome of the CD45 antibody, CFSE, wherein the cells responding to the stimulus are distinguished from the non-responding cells based on the positivity in relation to the activation markers. - isolating of vital immune cells from tissue using appropriate preparation techniques, for example, using enzymatic or mechanical extraction, - determining the total cell count, for example 10 4 to 1.5x10 6 cells are needed per stimulation batch, - prestaining of the isolated immune cells using a fluorochrome-coupled antibody which is aimed at the antigens expressed on the cells, e.g. CD45, or using fluorochromes capable of staining vital cells, e.g. carboxyfluorescein diacetate succinimidyl ester, CFDA-SE, - removal of unbound antibodies or fluorochromes by washing steps, - mixing of the prestained immune cells with heparinized whole blood of the same individual patient, - stimulating the mixed samples with the interesting antigen, with or without addition of co-stimulatory antibodies anti-CD49d and anti-CD28, anti-CD49d alone or anti-CD28 alone, - incubation for 30min to 4h at 35 to 39°C and 3 to 10% CO 2, - adding of a secretion inhibitor, e.g. brefeldin A, monensin or others, - incubation for 2 to 12 hours at 35 to 39°C and 3 to 10% CO 2, - lysis of erythrocytes and fixation of the leucocytes, - washing steps for isolating the leucocytes, - increasing cell membrane permeability by addition of a cell membrane permeability increasing substance, e.g. saponin, - incubation with specific fluorochrome-marked antibodies against activation markers, e.g. anti-CD69, anti-IFN-y, for at least 10min on ice or at room temperature or at 35 to 39°C, - repeated washing steps for removing unbound antibodies, - analysis of cells on a flow cytometry device and discriminating the immune cells from tissue against those from blood based on the fluorescence signal which was used for the prestaining, e.g. fluorochrome of the CD45 antibody, CFSE, wherein the cells responding to the stimulus are distinguished from the non-responding cells based on the positivity in relation to the activation markers.