To test this, IgE immune complexes or antigen alone were administered intranasally to antigen-sensitized mice (Determine 1). Scientific, Rockford, USA) in 200 l sterile PBS. Day 17C19 mice were anesthesized with 50 l answer consisting of Hypnorm (Vetapharma Ltd, Leeds, UK) and Dormicum (Roche, Basel, Switzerland) given subcutaneously. This answer was prepared by diluting Hypnorm 11 with sterile water and Dormicum 11 with sterile water separately and then mixing them together (11). Anesthesized mice were given daily intranasal doses with either 70 g OVA-TNP alone in sterile PBS or together with 70 g IgE-anti-TNP (Physique 1). Both solutions were prepared in a total volume of 30 l. In some experiments OVA sensitized mice were instead challenged with 1% OVA in PBS as GDC-0152 aerosol using a PARI nebulizer (Starnberg, Germany) for 30 min daily during day 17C19. Open in a separate window Physique 1 An outline of the experimental protocol.All mice were sensitized day 0 and day 7 with i.p. injections of 10 g OVA adsorbed to 1 1 mg alum. Day 17C19 the mice were challenged intranasally (i.n.) with either 70 g OVA-TNP alone or together with 70 g IgE-anti-TNP. Mice were analyzed 24 h after challenge GDC-0152 (see materials and methods). Preparation of mononuclear cells and mast cell progenitor quantification On day 20, mice were killed by an overdose of isoflurane (Schering Plough A/S, Farum, Denmark) and the SVIL lungs were perfused with 10 ml of sterile PBS administered via the right ventricle. Lungs and spleens were harvested into 10 ml of complete RPMI (RPMI 1640 made up of 100 U/ml penicillin, 100 g/ml streptomycin, 10 g/ml gentamicin, 2 mM L-glutamine, 10 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 50 M 2-mercaptoethanol and 10% heat-inactivated FCS (all from Sigma-Aldrich)). In one experiment, the mice were anesthetized and bronchoalveolar lavage was performed by flushing the lungs twice with 0.5 ml PBS. The obtained lungs were finely chopped with scalpels and transferred to 50 ml plastic tubes with 10 ml of complete RPMI 1640 plus 1 ml of 1830 models of collagenase type IV (Gibco, Paisley, Scotland, UK or Worthington, Lakewood, NJ, USA). The lung homogenates were incubated for 20 min at 37C. The released cells were harvested whereas the undigested tissue pieces were subjected to another enzymatic digestion (for a total of three digestions). The cells from the three digestions were pelleted, resuspended in 44% Percoll (Sigma-Aldrich) and underlayed with a 67% Percoll layer. After spinning at 400 for 20 min at 4C, lung mononuclear cells (MNC) were harvested from the 44/67% Percoll interface. The MNC obtained from two Percoll gradients per mouse lung were pooled and washed in complete RPMI 1640. The total number GDC-0152 of viable MNC was determined by trypan blue dye exclusion on a hemacytometer. The cells were serially 2-fold diluted to eight concentrations beginning at 20,000 cells/well in complete RPMI and 100 l of each dilution was added to eight wells of two sterile 96-well flat-bottomed tissue culture plates per mouse lung. Thereafter, each well received 100 l of gamma-irradiated (30 Gy) splenic feeder cells plus IL-3 (40 ng/ml) and SCF (40 ng/ml). Murine IL-3 was obtained as supernatants from the X-63 B-cell line [22] and quantified by ELISA. Recombinant SCF was purchased from Peprotech (#250-03, Rocky Hill, NJ, USA). After 10C12 days of incubation in humidified 37C incubators with 5% CO2, wells made up of mast cell colonies were counted with an inverted microscope. After culture, only mast cell colonies appeared in the plates. They were distinguished from the feeder matrix as small-medium sized round cells in large colonies [5], [23], [24]. The total number of lung MCp/mouse was GDC-0152 derived by multiplying the concentration of MCp (MCp/106 MNC) by the total number of MNC obtained from each mouse lung. Flow cytometry Lung MNC or cells from bronchoalveolar lavage were washed in FACS buffer (2% FCS in PBS pH 7.4) and fluorescence staining was performed at 4C in 100 l FACS buffer for 30 min. After pre-incubation with Fc-block (2.4G2, GDC-0152 BD Bioscience, Franklin Lakes, NJ, USA), the following antibodies were used to detect CD3+ and CD3+/CD4+ T cells and CD19+ B cells: Alexa 488 conjugated hamster anti-mouse CD3 (500-A2, Caltag Laboratories, Caltag-Medsystems Ltd, Buckingham, UK), PE conjugated rat.