Stimulated eosinophils were analyzed by flow cytometry. Supernatants were harvested, and secreted cytokines were quantified by using Bio-plex assay (Bio-Rad). To determine whether eosinophils support plasma cell survival, 6 days of secondary immunization eosinophils were isolated from the BM and plasma cells from the spleen. Using an isolation kit (Miltenyi Biotec), the purity of CD138+ splenic plasma cells was more than 90%. Cultures were set up as described previously 9. Briefly, 100 plasma cells were co-cultured together with eosinophils in U-bottomed 96-well plates for 24 h. After transfer of cells to anti-mouse Ig-coated
plates and further Roxadustat incubation at 37°C for 24 h, wells were washed and incubated with secondary antibody (Southern Biotech). The number of spots was counted by ELISPOT. To determine the viability of plasma cells, 104 plasma cells were co-cultured together with eosinophils isolated from BM of naïve, late primary (late 1°) or early secondary (late 2°) immunized mice for 48 h, and the percentage of living plasma cells was measured by staining with Annexin-V and PI. For cytospins, 1×105 sorted find more BM eosinophils in 150 μL complete medium were deposited into 24-well plates (Costar) fitted with cover slips. After
3 h, plates were centrifuged, washed with PBS and cells fixed with 100% cold ethanol for 10 min. Slides were stained with Alexa-546 conjugated monoclonal rat anti-MBP-specific antibody 28 together with rabbit anti-APRIL (Stressgen) or digoxigenin-conjugated monoclonal rat anti-IL-6-specific antibodies. Alexa-647-conjugated
Ergoloid goat anti-rabbit IgG (Invitrogen) and Cy5-conjugated anti-digoxigenin (DRFZ) antibodies were used as secondary antibodies. Staining was controlled by using rabbit IgG and rat IgG1 antibodies. Total RNA was extracted from 5×105 BM eosinophils using NucleoSpin®RNA II (Macherey-Nagel) according to the manufacturer’s instruction. Total RNA was reverse transcribed into cDNA using a Sensiscript RT kit (Qiagen). The levels of IL-4, APRIL, IL-6, IL-10 and TNF-α expression were determined by RT-PCR and real-time PCR as previously described 9, 27, 29. Primer sequences (TibMolBiol) for the amplification of IL-4, IL-6, APRIL and β-actin were described previously 9. The primers 5′seq: 5′-ctgactggcatgaggatcagc and 3′seq: 5′-ggcttggcaacccaagtaacc were used to amplify IL-10, the primers 5′seq: 5′-ggccaccacgctcttctgtct and 3′seq: 5′-ccagctgctcctccacttggt to amplify TNF-α. Real-time PCR was performed with the LightCycler system (Roche Diagnostics) using the Light-Cycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics). Each sample was run in triplicate. Values were normalized against β-actin, and the expression level was determined as the relative unit (RU) in comparison to non-activated normal eosinophils. For statistical analysis, a paired two-tailed Student’s t-test and two-way ANOVA was performed.