Alexa fluor 647 protein labelling kit

Spleen cells were prepared in FACS buffer containing 50μg/ml gentamicin for inhibiting extracellular S. Typhimurium from entering cells during preparation. Cells were stained for surface markers and then fixed and permeabilised using the FoxP3 staining kit (eBioscience) according to manufacturer’s instructions. Monoclonal S. Typhimurium LPS (clone 1E6, Thermo Fisher) was conjugated using the Alexa Fluor 647 Protein Labelling Kit (Thermo Fisher) and used for intracellular labelling of S. Typhimurium. We have verified that the antibody stained all three strains of S. Typhimurium used in this study, but does not cross-react with E. coli.

Purified rNM23-H1, rNDPK form S.pneumoniae and Bovine serum albumin (BSA) were diluted to 2mg/ml and dialyzed at 4°C overnight in 2000 volumes of 0.5M NaCl, 0.1M imidazole in phosphate buffered saline (PBS), pH 8.2, prior to labelling using the Alexa Fluor 647 Protein Labelling Kit (ThermoFisher Scientific) according to manufacturer’s instructions.