Red blood cells lysis buffer

Blood samples were taken from the temporal plexus of mice 0, 12, and 28 days after priming and 3 and 10 days after boosting. Samples were incubated for 30 min at 37°C and centrifuged at 1,200 × g at 4°C for 10 min for collecting sera that were stored at −80°C. Draining lymph nodes (sub iliac, medial, and external) and spleens were collected 7 and 28 days after priming, and 3 or 10 days after boosting. Samples were mashed onto 70 µm nylon screens (Sefar Italia, Italy) and washed two times in complete RPMI medium [RPMI (Lonza, Belgium), 100 U/ml penicillin/streptomycin, and 10% fetal bovine serum (Gibco, USA)]. Samples were treated with red blood cells lysis buffer (eBioscience, USA) and counted with cell counter (Bio-Rad).

Bone-marrow-derived DC were cultured with GM-CSF as described previously [35 (link)]. AM and RAW cells were cultured as described previously [36 (link)]. Ten week-old mice were euthanized, their rib cage removed and small incision was cut in the upper part of trachea. Lungs were washed at least 10 times by 1 ml of phosphate buffered saline (PBS) + 2% fetal calf serum (FCS) + 2 mM EDTA. The washing medium containing cells was kept on ice in 50 ml falcon tubes containing 10 ml of AM medium (Roswell Park Memorial Institute medium (RPMI) + 10% FCS + 1% pen/strep + 1% pyruvate + 1% glutamine). Cells were then centrifuged at 1500 rpm, 4°C for 5 min and red blood cells lysis was performed on ice using red blood cells lysis buffer (eBioscience, 00-4333-57) according to the manufacturer's instructions. Cells were then resuspended in AM medium and seeded in uncoated 6-well plates (Thermo Scientific, 150239). Cells were grown in AM medium with media supplemented with 2.5% GM-CSF. HeLa cells were maintained in Dulbecco's modified eagle medium (Gibco Invitrogen) supplemented with 10% FCS (Hyclone, PERBIO), at 37°C and 5% CO₂.

Cells were harvested from whole blood samples and FACS was performed using 3 fluorochrome-conjugated antibodies: i) fluorescein-conjugated anti-CD4 (R&D, #967211) ii) Rα PE-conjugated anti-CD25 (R&D, #968436) iii) Alexa Fluor-conjugated anti-FoxP3 (R&D, #968435). Beforehand, a red blood cells lysis buffer (eBioscience, #00433357) was used to remove Red Blood cells from the whole blood samples. A FoxP3/transcription factor permeabilization buffer (R&D, #43481) was used for nuclear permeabilization before incubation with anti-FoxP3 antibody. Flow cytometry was performed on an FACS Canto II flow cytometer to evaluate cell fluorescence. Obtained data were analyzed using FACS Diva software (BD, San Jose, CA). Percentages of FoxP3+/CD4+CD25+ cells and of FoxP3+/CD4+ total cells were calculated. Fluorescence Minus One Control was defined for FoxP3 using an isotype control. Gating strategy is given in Supplementary Figure 2.