Dnp klh

Cells were incubated overnight at 37°C with anti-DNP IgE hybridoma supernatant at a final IgE concentration of 0.5 µg/ml. Cells were washed once with RPMI, then with Tyrode buffer (10 mM HEPES pH 7.4, 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.01% BSA). Cells were activated in Tyrode buffer with 100 ng/ml of DNP-KLH (keyhole limpet hemocyanin conjugated DNP, Sigma-Aldrich) at 37°C in the dark, for 45 minutes. Cells were subsequently washed in ice-cold Tyrode buffer.
For the Annexin V-APC (Becton Dickinson Biosciences, San Jose, CA, USA) staining, 100 µl Annexin V-APC were added to 2×10⁶ cells (in 500 µl), placed 30 min on ice in the dark. The cells were then labeled with 20 µg/ml Propidium Iodide 3 minutes prior to FACS analysis. Analysis and sorting by flow cytometry were performed using FACS Aria cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). For plasmid library selection, 2×10⁸ and 10⁷ cells were sorted at the first round and following rounds respectively. For retroviral selection, 4×10⁷ and 10⁷ cells were sorted at the first round and following rounds respectively.

Non-infected and L. sigmodontis-infected mice were vaccinated at indicated time points post infection by i.p. injection of either 100 µg alum-precipitated dinitrophenol-keyhole limpet hemocyanin (DNP-KLH, Sigma-Aldrich, Munich, Germany), 100 µg 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP) conjugated to Ficoll (NIP-Ficoll) (Biosearch Technologies, Navato, USA), or by s.c. injection of 30 µg alum-precipitated DNP-KLH into the hind footpad. For analysis of serum antibodies, blood was collected from mice by submandibular bleeding of the facial vein 7, 14 and 21 days after vaccination and allowed to coagulate for 1 h at RT. Serum was collected after centrifugation at 10,000× g for 10 min at RT and stored at −20°C for further analysis. For analysis of spatial separated cellular responses, mice were sacrificed at the indicated time point, and spleen and popliteal lymph nodes (popLN) were dissected. A total of 2.5×10⁵ splenocytes or popLN cells were cultured in 3–5 replicates in 96-well round-bottom plates in RPMI 1640 medium supplemented with 10% FCS, 20 mM HEPES, 2 mM L-glutamine and gentamicin (50 µg/mL) at 37°C and 5% CO₂. The supernatant was harvested after 21 days of culture and DNP- and L. sigmodontis-specific IgG1, IgG2a and IgG2b were quantified by ELISA.