Flat bottomed 48 well plate

To analyse the T helper cell‐specific cytokine production, 500 000 live PBMCs were stimulated for 48 hours (at 37°C and 5% CO₂) with Dynabeads^TM Human T‐activator CD3:CD28 (Thermo Fisher Scientific, cat‐nr: 11161D) at a 2:1 cell:bead ratio.
In order to analyse the IgE and IgG4‐ab secreting B cells, 500 000 live PBMCs were stimulated for 5 days with, respectively, a combination of CD40L (1 μg/mL) and IL4 (10 ng/mL) or R848 (1 μg/mL) and IL2 (10 ng/mL) (all from Mabtech AB, Nacka, Sweden, cat‐nr's: 3810‐2H, 3854‐2A).
Cell culture supernatants were collected from all stimulations and stored at −20°C for up till a month until further use. All stimulations were performed in a flat‐bottomed 48‐well plate (Costar, cat‐nr: 10065370).

Frozen CBMCs and PBMCs were thawed and washed with RPMI‐1640 supplemented with 20 mm HEPES (GE Healthcare – HyClone Laboratories). The cells were counted and viability was assessed with Trypan Blue staining; only cells with sufficient viability were used for the functional assays. Subsequently, the cells were resuspended in a concentration of 10⁶ cells mL⁻¹ in cell culture medium, consisting of RPMI‐1640 supplemented with 20 mm HEPES, 100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin, 2 mm l‐glutamate (2 mm) (all GE Healthcare – HyClone Laboratories) and 10% heat‐inactivated fetal calf serum (Sigma Aldrich). The cells were either rested for a minimum of 1 h before basal phenotypic staining or stimulated for 24 h with 40 ng mL⁻¹ HMB‐PP (Sigma Aldrich) or Dynabeads Human T‐activator CD3:CD28 (Thermo Fisher Scientific, Waltham, MA, USA) at a 2:1 cell‐to‐bead ratio at 37°C and 5% CO₂ in a flat‐bottomed 48‐well plate (Costar, Cambridge, UK). Brefeldin A (BD Biosciences) was added during the last 4 h of incubation.