The degrees of APRIL expression were normalized to those of trout EF-1 and expression levels calculated using the 2 2?Ct method, where Ct is determined by subtracting the EF-1 value from the target Ct as described previously (26, 27)

The degrees of APRIL expression were normalized to those of trout EF-1 and expression levels calculated using the 2 2?Ct method, where Ct is determined by subtracting the EF-1 value from the target Ct as described previously (26, 27). in the number of mature B cells as the B cell development in these animals seems normal (16). These results suggest that, for 30?min at 4C) of diluted blood on 51% continuous Percoll (GE Healthcare) density gradients. A transcardial perfusion of the rainbow trout was performed using Ringer answer pH 7.4 containing 0.1% procaine to remove blood from fish tissues. Adipose tissue, gonad, brain, foregut, stomach, pyloric caeca, midgut, hindgut, heart, spleen, skin, gills, anterior and posterior kidney, Biotin-PEG3-amine liver, and thymus samples were then collected and placed in Trizol (Thermo Fisher Scientific). DNase I-treated total RNA was prepared from tissue samples or PBLs using a combination of Trizol (Invitrogen) and an RNAeasy Mini kit (Qiagen) as described previously (25). Total RNA was eluted from the columns in RNase-free water, quantified using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific) and stored at ?80C until use. For each sample, 2?g of total RNA was reverse transcribed using Bioscript reverse transcriptase (Bioline Reagents Ltd.) primed with oligo (dT)12C18 (0.5?g/ml), following the manufacturers instructions. cDNA was diluted in nuclease-free water and stored at ?20C. To evaluate the levels of APRIL transcription, real-time PCR was performed in a LightCycler 96 System instrument (Roche) using FastStart Essential DNA Green Grasp reagents (Roche) and Biotin-PEG3-amine specific primers (Table S1 in Supplementary Material) as previously described (23). Each sample was measured in duplicate under the following conditions: 10?min at 95C, followed by 40 amplification cycles (30?s at 95C and 1?min at 60C). The levels of APRIL expression were normalized to those of trout EF-1 and expression levels calculated using the 2 2?Ct method, where Ct is determined by subtracting the EF-1 value from the target Ct as described previously (26, 27). Unfavorable controls with no template and reverse transcriptase controls [?room heat (RT)] were included in all experiments. Transcriptional Analysis of Isolated Leukocyte Populations Single cell suspensions from spleen and gills were prepared using 100-m nylon cell strainers (BD Biosciences) and L-15 medium supplemented with antibiotics (P/S) and 5% FCS. Skin cell suspensions were also prepared. For this, prior to cell extraction, pieces of LHR2A antibody skin were incubated for 30?min at 4C in L-15 medium with antibiotics (P/S) and 5% FCS, followed by agitation for 30?min in PBS containing 1?mM EDTA and 1?mM DTT. Tissue digestion was performed using 0.15?mg/ml collagenase type IV from (Sigma) in L-15 for 1.5?h at 20C. All cell suspensions were placed onto 30/51% Percoll density gradients and centrifuged at 500??for 30?min at 4C. Cells at the interface were collected and washed twice in L-15 medium made up of 5% FCS. The constitutive levels of APRIL transcription were studied in IgM+ B cells and T cells from spleen as well as from CD8+ dendritic cells (CD8+ Biotin-PEG3-amine DCs) found in skin and gills after isolating the cells following the Biotin-PEG3-amine methods previously established (23, 28). The expression levels of Blimp-1, CD80/86, CD83, and CD40 were also analyzed on IgM+ B cells from spleen using specific primers previously described (Table S1 in Supplementary Material). For this, DNase I-treated total RNA was reverse transcribed directly from FACS sorted populations using the Power Sybr Green Cells-to-Ct Kit (Invitrogen) following the manufacturers instructions. For comparative purposes, RNA was also isolated from the RTS11 rainbow trout macrophageCmonocyte cell line (29). Real-time PCR was performed using SYBR Green PCR core Reagents (Applied Biosystems) using specific primers and following the manufacturers instructions as described previously (30). APRIL Transcription at Early Life Stages To investigate whether APRIL is usually expressed at early life stages, eyed eggs at different degree days (DD) post-fertilization (~306 DD, ~354 DD, and ~402 DD), immediate post hatch fry (hatch, ~450 Biotin-PEG3-amine DD), pre-first feeding fry (PFF, ~562 DD), fry at the stage of full disappearance of the yolk sac (first feeding, FF, ~674 DD), and fry 3?weeks following first feeding (Fry, 786 DD) were sampled. The fish were maintained at 16C in recirculated freshwater. Total RNA was extracted and cDNA prepared for real-time PCR analysis from eggs or whole fry using a combination of Trizol (Invitrogen) and an RNAeasy Mini kit (Qiagen) as described above for the analysis of.