Anti goat igg pe cy7

ICS was performed using the Cytofix/cytoperm kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. A total of 1 × 10⁶ freshly isolated splenocytes were stimulated with 1 μM of an individual peptide or inactivated virus in the presence of 1 μg/mL brefeldin A (Sigma-Aldrich, Dorset, UK) in 1000 μL of culture medium at 37 °C for five hours [12 (link)]. Cultures were stimulated with UV-inactivated RSV Long strain, F, NP, and M2-1 peptides as shown in Table 1. F peptides are recognition sites of palivizumab (synagis®). NP_306–314 is based on the human experiments and the others are on mouse. After the stimulation, splenocytes were washed and incubated with an anti-CD8 antibody (R&D Systems, Minneapolis, MN, USA) at 4 °C for 30 min. Splenocytes were fixed with fixation buffer in the Cytofix/cytoperm kit, and intracellular cytokines were stained with a goat IgG antibody against IFN-γ (R&D Systems, USA) and anti-goat IgG PE-Cy7 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4 °C for 60 min. Cellular populations were analyzed using flow cytometry with Cytomics FC 500 (Beckman Coulter, Inc., Indianapolis, IN, USA) and counted until 100,000 cells.

A total of 1–2×10⁶ freshly isolated splenocytes was stimulated with 1 μM of individual peptides in the presence of 10 μg/ml brefeldin A (Alomone labs, Israel) in 500 μl culture medium (RPMI, supplemented with 10% FCS) at 37°C for 5 hours. Peptides corresponding to CD8⁺ T cell epitopes were synthesized (Medical … Biological Laboratories Co. Ltd. Japan) [13 (link), 14 (link)]. After the stimulation with 1 μM of respective peptides, cells were harvested, washed, and incubated with an anti-CD8 antibody (R&D Systems, USA) for 30 min. Cells were fixed with the Cytofix/cytoperm kit (BD Pharmingen, San Diego, CA), and intracellular cytokines were stained with a goat IgG antibody against cotton rat IFN-γ (R&D Systems, USA) and anti-goat IgG-PE-Cy7 (Santa Cruz Biotechnology, Inc., USA) for 60 min. Cellular populations were analyzed using flow cytometry with Cytomics FC 500 (Beckman Coulter, Inc., USA) [15 (link)]. The splenocytes were counted 100,000 cells or for six minutes, and CD8⁺ IFN-γ⁺ cells were analyzed in gate.