Tnp lps

For tamoxifen treatment, mice were injected intraperitoneally with tamoxifen (1 mg per mouse) in corn oil daily for four consecutive days and analyzed 8 days after the last injection. For experiments involving an immune challenge, chimeras were given tamoxifen (1 mg per mouse) in corn oil daily for four consecutive days via oral gavage and challenged with antigens or influenza at day 8 after the last tamoxifen administration. For NP-OVA immunization experiments, antigen for immunization was prepared by mixing NP₂₀-OVA (20 molecules of NP linked to OVA; Biosearch Technologies), 10% KAL(SO₄)₂ dissolved in PBS at a ratio of 1:1, in the presence of LPS (Escherichia coli strain 055:B5; Sigma) at pH 7. Mice were immunized intraperitoneally (100 μg NP-OVA and 10 μg LPS) for analysis of NP-specific antibody response. Nine days after immunization, sera, spleens and mesenteric lymph nodes were collected from the mice. Thirteen days after infection, spleens, mediastinal lymph nodes and lungs from the mice were harvested for analysis. For T-independent immune response, mice were given 50 μg TNP-LPS (Biosearch Technologies) intraperitoneally. Sera and spleens were collected on day 7 after immunization.

Rainbow trout of approximately 10-15 cm received 50 µg of 2,4,6-Trinitrophenyl hapten conjugated to lipopolysaccharide (TNP-LPS) (Biosearch technologies) in 200 µl of sterile saline solution (0.9% sodium chloride, ClNa) by means of an intraperitoneal injection. The conjugated LPS was the high molecular weight form of Escherichia coli with repeating polysaccharide O-chain. A mock-immunized group (control) received an intraperitoneal injection of 200 µl of sterile saline solution. Sampling was performed after 7, 14 and 28 days, collecting 8 rainbow trout from each group. After sacrificing the rainbow trout by benzocaine overdose, leukocytes were isolated from spleen, head kidney, peritoneal cavity, peripheral blood and AT as described above to quantify the number of total and TNP-specific trout IgM-secreting cells by ELISpot and to perform flow cytometry analysis of the IgM⁺ B cell population.
For immunofluorescence staining, the AT from 6 rainbow trout from each group were collected at day 21 post-immunization. The collected tissues were fixed in 4% paraformaldehyde for 24 h and processed for paraffin embedding following routine histological procedures, as previously described (27 (link)).

Blood (10 mL) was taken by venipuncture from healthy volunteers and B-cells were isolated and immortalized as described by Tosato et al. [9 (link)]. Immortalized cells were cultured in tissue culture plates for 6 days and then stimulated by 40 μg/ml TNP-LPS (Biosearch Technologies), 10 U/mL hIl-1 (BD) and 100 U/ml hIl-2 (BD). The supernatant of each well was collected and used in an opsonophagocytic killing assay (OPA) against E. faecalis 12030 to identify the well resulting in the highest killing. B-cells from this well were distributed into a new tissue culture plate. Supernatants were again tested by OPA and the cells of the well leading to the highest killing were distributed into a new plate. After 4 rounds, B-cells in the wells with the strongest response were lyzed and mRNA and cDNA was prepared.