Cytokine profile of T cells obtained with the same reagents but different fixation time and temperature.

(A) PBMCs were fixed with PP for 30 minutes at 4°C (left panels) or 5 minutes at 37°C (right panels) and then intracellular stained. Quadrant settings were based on unstained negative controls. From top to bottom: unstained cells; cells stained with anti–IL-4 FITC, anti–IFN-γ PE (Pharmingen, Palo Alto, CA) and anti-CD3 PercP (B&D, San Jose, CA); cells incubated with 25-fold excess of unlabeled anti–IFN-γ and stained as above; cells incubated with 25-fold excess of unlabeled anti–IL-4 and stained as above. The data was collected in a FACScan and analyzed using CellQuest software (B&D, San Jose, CA). Data gated on the CD3⁺ population are shown. (B) Comparative analysis of the 2 techniques used on PBMCs obtained before (PBMC) and after (G-PBMC) G-CSF treatment. The data represent mean ± SD obtained from 7 (30 minutes at 4°C ) or 5 (5 minutes at 37°C) different stem cell donors for each technique shown.