Phytohemoagglutinin pha

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood samples (10 U/mL heparin) by Ficoll-Hypaque density gradient centrifugation (Lymphoflot, Biotest, Germany). PBMC were then washed with phosphate-buffered saline (PBS) and either utilized for membrane antigens assessment or cultured up to 72 h in 96-well U-bottomed microplates (Becton Dickinson, CA, USA) with RPMI-1640 complete medium (Biochrom AG, Germany) supplemented with 10% autologous plasma at 37 °C in a 5% CO 2 atmosphere in humified air. PBMC were cultured in the absence (controls) or in the presence of the following stimuli or allergens (kindly provided by Anallergo, Florence, Italy): phytohemoagglutinin (PHA, 10 μg/ml, Sigma Aldrich, Italy), lipopolisaccaride (LPS, 20 μg/ ml, Sigma Aldrich, Italy), phorbol-myristate-acetate (PMA, 20 μg/ ml, Sigma Aldrich, Italy) + ionomycin (1 μM, Sigma Aldrich, Italy), Olea (1 μg/ml), Parietaria (1 μg/ml), Graminaceae (1 μg/ml), and Dermatophagoides (2.5 μg/ml). At the end of the incubation period, supernatants were collected and kept frozen at -30°C until sHLA-G determination and cells were washed with PBS and used for membrane antigens assessment.