Anti gm csf efluor660 gm2f3

A portion of each tonsil was collected in dry conditions, immediately frozen in liquid nitrogen, and stored at -80°C until processing. Tonsil cryosections (8-μm thick) were obtained with the cryo-microtome CM1950 (Leica), fixed with 4% formaldehyde for 10 min at RT and washed three times with PBS. Sections were blocked with 1 mg/ml of human immunoglobulins (Gentaur) for 30 min at RT, washed with PBS and stained with an antibody mix containing anti-human CD19-V450 HIB19 (BD Pharmingen), anti-CD3-PE SK7 (BD bioscience), anti-GM-CSF-eFluor660 GM2F3 (eBioscience), anti-IgD-BrillantViolet421 IA6-2 (Biolegend), all diluted in 1 mg/ml human immunoglobulins for 1 h at RT. After incubation, sections were washed again with PBS and mounted with a coverslip using Gold anti-Fade reagent (Life Technologies) for analysis by confocal microscopy.

5 x 10⁵ (300 μl/well) tonsil-derived cells were seeded on a poly-L-lysine-coated coverslip in a 24-well plate. Cells were incubated for 2 h at 37°C or 4°C with or without heat-inactivated S. aureus or PhRodo Green S. aureus Bioparticles at a MOI of 10:1 (bacteria:cells). After incubation, cells were fixed with 2% formaldehyde for 30 min at RT, washed with PBS and permeabilized with 0.1% Triton X-100. Coverslips were then washed again with PBS and blocked with 3% BSA—10% goat serum (Invitrogen) for 20 min at RT. Finally, coverslips were washed with PBS and incubated for 30 min at RT with antibody mix containing human anti-CD19-V450 HIB19 (BD Pharmingen), anti-GM-CSF-eFluor660 GM2F3, anti-MPO-PE 455-8E6 (eBioscience), all diluted in 0.1% BSA + 1% goat serum. After incubation, coverslips were washed and mounted on a slide with Gold Anti-Fade reagent (Life Technologies) for analysis by confocal microscopy.