Experimental conditions for measuring MHCII-specific cellular fluorescence by flow cytometry. Tumor cells were removed from plastic with 0.25% trypsin and 0.53 mM EDTA, washed, stained with FITC-conjugated monoclonal antibodies (mAb) and analysed on a FACSCalibur™ flow cytometer. Regions R1, R2 and R3 were drawn to exclude debris (G), dead cells (A, B, C) and cellular aggregates (D, E, F). Panels A and D show cells stained with propidium iodide alone. Panels B and E show cells stained with propidium iodide and the isotype-matched control mAb (IgG2a-FITC, 1.0 μg/50 μl). Panel C and F show cells stained with propidium iodide and the mAb against human HLA-DR,DP,DQ (anti-MHCII-FITC, clone Tü39, 0.25 μg/50 μl). Panel H shows frequency distributions of cells that passed R1 & R2 & R3 logical gate. M1, M2 and M3 are the median values of autofluorescence peak (M1 = 222), isotype control peak (M2 = 234) and HLA-DR peak (M3 = 445).