To confirm that BAFF binding to B cells was specific, we blocked the union having a polyclonal antibody raised against recombinant trout BAFF in mice. on B cells, upregulating the manifestation of membrane MHC II, improving the survival of fish na?ve B cells and antibody-secreting cells, and increasing the secretion of IgM. Remarkably, we also demonstrate that BAFF isn’t just produced in fish by myeloid cells but is also produced by a subset of splenic B cells. Therefore, if this B cell-produced BAFF shows to be actively regulating this same B cell subset, our findings point to an ancient mechanism to control B cell differentiation and survival in lower vertebrates, which has been silenced in mammals in physiological conditions, but reemerges under pathological conditions, such as B cell lymphomas and autoimmune diseases. for 30?min at 4C. The SRSF2 interface cells were collected and washed twice in L-15 comprising 5% FCS. When required, leukocytes were incubated in the presence of TNP-LPS (Biotools) at a final concentration of 5?g/ml and/or recombinant rainbow trout BAFF at a final concentration of 3?g/ml. A wide range of doses of these stimuli were tested and the optimal doses were selected based on their effect on B cell survival (data not demonstrated). Circulation Cytometry The anti-trout IgD [mAb mouse IgG1 coupled to R-phycoerythrin (R-PE), 5?g/ml], the anti-trout IgM [1.14 mAb mouse IgG1 coupled to fluorescein (FITC) or to allophycocyanin (APC), 1?g/ml], and the anti-trout MHC II -chain (mAb mouse IgG1 coupled to APC, 2?g/ml) used in this study have been previously characterized (43, 44). All the mAbs were fluorescently labeled using fluorescein, R-PE, or APC Lightning-Link K252a labeling packages (InnovaBiosciences), following manufacturers instructions. Spleen leukocytes were incubated with specific antibodies for 30?min in the case of anti-IgM or anti-MHC, or 45?min in the case of anti-IgD, washed three times with staining buffer (PBS containing 1% FCS and 0.5% sodium azide), and analyzed. K252a A biotinylated version of anti-BAFF (pAb mouse IgG, 1?g/ml) was used to determine endogenous BAFF manifestation by leukocytes. To carry this out, cells were incubated for 30?min with biotinylated anti-BAFF pAb, then washed three times with staining buffer, and incubated for another 30?min with streptavidin-FITC (Thermo Fisher Scientific). In all cases, isotype settings for mouse mAbs and anti-BAFF pAb (BD Biosciences) were tested in parallel to discard unspecific binding of the Abs. All the incubations were performed at 4C. After incubation with the related stimuli, samples were incubated with 10?g/ml propidium iodide (Thermo Fisher) for 5?min in the dark, and cell viability was analyzed in our experimental conditions (for 10?min at 4C) over a cushioning of 3% (excess weight/volume) bovine serum albumin [(BSA), Fisher Scientific] in PBS supplemented with 4.5% (weight/volume) d-glucose (Sigma). Cells were resuspended in staining buffer, labeled with K252a an anti-IgM mAb fluorescently labeled with FITC, and analyzed on a FACSCalibur circulation cytometer equipped with CellQuest sofware (BD Biosciences). The analysis was also performed with FlowJo 10. B Cell Proliferation The BrdU Circulation Kit (Becton Dickinson) was used to measure the proliferation of IgM+ B cells in response to BAFF and/or TNP-LPS following manufacturers instructions. Splenocytes at a concentration of 2??106 cells/ml were incubated for 3?days at 20C with the stimuli while described above. Bromodeoxyuridine (BrdU, 10?M) was then added to the cultures and the cells were incubated for an additional 24?h. The cells were collected and stained with APC-anti-IgM mAb (1?g/ml). Briefly, to analyze incorporation of BrdU, cells were then fixed and permeabilized with Cytofix/Cytoperm Buffer for 15?min on snow, in that case incubated with Cytoperm Permeabilization Buffer In addition for 10?min on snow, and re-fixed with Cytofix/Cytoperm Buffer for 5?min at RT. Cells were then incubated with DNase (30?g/106 cells) for 1?h at 37C to expose the incorporated BrdU. Finally, cells were stained with FITC anti-BrdU antibody for 20?min at RT and analyzed by circulation cytometry. Confocal Microscopy Splenocytes were obtained as explained above. To establish BAFF binding to trout IgM+ B cells, leukocytes were incubated with 3?g/ml of recombinant BAFF in L-15 press supplemented with 5% FCS. After 1?h at 20C, the cells were washed with serum-free L-15 medium, seeded about poly l-lysine coated slides, and incubated at 20C for 30?min. After softly washing with PBS, the slides were fixed in 4% PFA.