Cells were left in culture for 4 days in 5% CO2 at 40 °C. At day 4, EDTA (20 mm) was added to all wells to a final concentration of 2 mm EDTA. The plate was left for 10 min in the CO2 incubator at 40 °C to detach cells from the well. Finally, each sample was mixed by carefully pipetting up and down before transferring

it to FACS tubes. Flow cytometry.  Staining was carried out in tubes by adding 110 μl cell suspension to 90 μl of FACS buffer (0.2% BSA, 0.2% sodium azide, 0.05% normal horse serum in PBS) containing CD4-RPE (Clone CT4) and CD8α-APC (Clone CT8 or Clone 3-298) or CD8α-RPE (Clone EP72 or Clone 3-298). All antibodies were purchased from SouthernBiotech (Birmingham, AL, selleckchem USA). In addition, propidium iodide (Fluka BioChemica, Buchs, Switzerland) was added to exclude dead cells. Cells were incubated at 4 °C for 15 min and then washed once with 2 ml FACS buffer by centrifugation at 295 g for 5 min. All flow cytometry analyses were

performed on a BD FACSCanto™ (BD Biosciences, San Jose, CA, USA) equipped with a 488-nm blue laser and a 633-nm red laser. Using the FACSDiva software, we aimed at collecting a minimum of 10,000 live cells from each sample. Titration of all antibodies was performed prior to the experiment in order to determine the optimal staining concentrations, Midostaurin cost and the multicolour panel was carefully evaluated using fluorescence minus one (FMO) controls [15]. Statistical analysis.  Antigen-specific stimulations were run in triplicates with the exception of the experiment where the effect of anticoagulant and type of serum were tested, as this experiment was run in duplicates. For all optimization steps much and for experiment 1, the mean percentage of proliferated cells ± SE was calculated and shown. The CFSE proliferation data for experiment 2 were tested using standard anovaF-tests on a 5% significance level, with dose and breeding line as the classification variables. Normally, blood is stabilized with heparin, and FBS is used as an additive to growth medium

in cellular stimulation assays. We wanted to test EDTA as a substituent for heparin as anticoagulant in the blood samples and autologous serum from an NDV-vaccinated chicken (CIS) as a substituent for FBS in the cell culture medium used for our recall proliferation assay assessed for both CD4+ and CD8α+ (Fig. 1A) T cells. The strategy for gating on CD4+ and CD8α+ T cells was debris exclusion on the Forward Scatter (FSC) – Side Scatter (SSC) dot plot followed by exclusion of dead cells by PI staining. Out of the live cells, CD4 cells were gated positive at the PE axis and CD8α cells were gated positive at the APC axis in a PE-APC dot plot (Fig. 1A). Finally, the CD4+ and CD8α+ T cells were shown in a dot plot with CFSE on the x-axis, and the percentage of proliferated CD4+ and CD8α+ T cells were measured. Fig. 1B shows the results from one representative sample.