Spots representing single Ab-secreting cells were developed with the substrate (a buffered solution containing 4 mg of 4-chloro-1-naphthol, Sigma–Aldrich). The spots were counted with the aid of a dissecting microscope. Flow cytometry. Single cell suspensions (1 × 106/sample) of pooled NALT and NP from seven untreated and immunized mice were stained with fluorochrome-labelled mAbs as described previously . The surface phenotype of cells was analysed using anti-mouse mAb (Becton Dickinson Technologies, Gaithersburg, MD, USA) purchased from PharMingen (San Diego, CA, USA). The mAbs used in this study
included anti-CD45R/B220+ phycoerythrin (PE) (RA3-6B2), anti-CD3+ fluorescein isothiocyanate (FITC) (molecular complex 17A2), anti-CD3+ peridinin chlorophyll protein (PerCP) (CD3e chain) JAK pathway (145-2C11), anti-CD4+ FITC (L3T4) (6K1.5) and anti-CD8a+ PE (Ly-2) (53–6.7).
For analysis of activation marker expression, the mAb used were anti-CD25 (FITC) (IL-2Ra chain, p55) (7D4), anti-CD25 (PE) (IL-2Ra chain, p55) (7D4), anti-CD69 (FITC) (very early activation antigen) (H1.2F3) and anti-CD69 (PE) (H1.2F3). To perform flow cytometric analyses, relative fluorescence intensities were measured using a FACSCalibur cytometer (Becton Dickinson, San Jose, CA, USA) and BD cell quest Pro v.5.1.1 software (Becton Dickinson). For each phenotypic characteristic data were collected for 20,000 events. Lymphocyte phenotypes were determined by two or three colours immunofluorescence. The percentage of cells labelled with each mAb was calculated in comparison with cells RAD001 order stained with isotype control antibody. B and T-cells were analysed within
a lymphocyte gate defined by forward and side light Non-specific serine/threonine protein kinase scatter. Background staining was controlled by labelled isotype controls (Pharmingen) and never exceeded 1.0% of cells. The results represent the percentage of positively stained cells in the total cell population exceeding the background staining signal. Each analysis was performed at least three times for verification, and the data represent the mean ± standard deviation from three to five experiments using cells of a given tissue from seven mice. Detection of intracellular cytokines: IL-2, IFN-γ, IL-4, IL-5, IL-10 and TNF-α. The production of cytokines was measured through intracellular staining, and all the phenotypic assays of NALT and NP were performed in parallel as described in . Statistical analysis. The statistical analysis was performed using Mann–Whitney U-test, taking P < 0.05 as significant. The number of lymphocytes recovered from NALT following immunization of BALB/c mice with Cry1Ac was increased. The yield of lymphocytes from normal mice in NALT was 7.2 (±0.7) × 105 cells, while in the immunized group the number of cells obtained was 1.2 ± 0.3 × 106 cells (P < 0.05). The number of cells obtained from NP was slightly increased by immunization 1.4 (±0.