Ficoll diatrizoate gradient centrifugation

Heparinized blood was centrifuged at 400 × g for 10 min, plasma separated and stored at 4°C. Samples were refilled with RPMI-1640 and PBMC isolated by Ficoll diatrizoate gradient centrifugation (LSM; ICN Biomedicals). Cells were then washed, resuspended in freezing medium (RPMI 1640, 10% DMSO, 45% heat inactivated FCS, Harlan Bioproducts) and stored in liquid nitrogen until usage. For in vitro culture the cryopreserved cells were thawed gently, washed twice and cultured with RPMI 1640 medium supplemented with penicillin-streptomycin (100 U and 100 μg/ml, respectively), L-glutamine (2 mM), 1% NEAA MEM (all from PAN–Biotech, Aidenbach, Germany) and 5% heat inactivated AB human serum (Biochrom, Berlin, Germany). For stimulations and intracellular staining cells were counted using trypan blue and adjusted to 1 × 10⁷ cells/ml. 2 × 10⁶ cells/well were placed on round-bottom 96 well tissue culture plates, stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (both Sigma-Aldrich, MO, USA) at concentrations of 25 ng/ml and 0.5 μg/ml or media alone and incubated for 4 h at 37°C. Brefeldin A solution (10 μg/ml, eBioscience, CA, USA) was added after 30 min. After 4 h cells were washed, stained and analyzed as given below.

Heparinized blood was collected and PBMC isolated by Ficoll diatrizoate gradient centrifugation (LSM; ICN Biomedicals). Erythrocytes were lysed using ACK lysis buffer (Biosource). Cells were then washed and cultured in RPMI-1640 (BioWhittaker), supplemented with 20 mM glutamine (BioWhittaker), 10% heat-inactivated FCS (Harlan Bioproducts for Science), and 50 µg/ml of gentamicin (Mediatech). PBMC were cultured with BmA, Mf, PPD or PMA/ionomcyin in 24-well tissue culture plates (Corning) at concentrations of 5×10⁶/well. After 24 h, culture supernatants were collected and analyzed for cytokines.