Pe conjugated anti cd11b m1 70

Femoral and tibial bone marrow cells were harvested from mice, and subsequently cultured in RPMI-1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FBS, sodium pyruvate (Thermo Fisher Scientific), MEM NEAA (Thermo Fisher Scientific), and recombinant (r) M-CSF (40 ng/ml, BioLegend) for 6 days, with medium changes every 2 days. The differentiated BMDMs were treated with rIFN-γ (100 ng/ml; BioLegend) + LPS (50 ng/ml; L5273, Sigma-Aldrich) or rIL-4 (20 ng/ml; BioLegend) for 16 to 24 hours to achieve M1 or M2 polarisation, respectively. In order to evaluate M2 polarisation, we stained BMDMs treated with rIL-4 with PE-conjugated anti-CD11b (M1/70, BioLegend,), FITC-conjugated anti-F4/80 (BM8, eBioscience), and Alexa Fluor® 647-conjugated anti-CD206 (MR5D3, AbD Serotec), and analysed on a FACSCalibur™ cytometer (BD Biosciences).

Single-cell spleen suspensions obtained from WT and TG mice were prepared by treatment with red blood cell lysis solution (0.15 M NH₄Cl and 0.1 mM Na₂EDTA) for 30 min at room temperature. Fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (M1/70, Biolegend, CA, USA), FITC-anti-CD4 (GK1.5, BD Biosciences, CA, USA), FITC-anti-F4/80 (BM8, Thermo Fisher, MA, USA), phycoerythrin (PE)-conjugated anti-CD11b (M1/70, Biolegend), PE-conjugated anti-Ly6G (RB6-8C5, Thermo Fisher), APC-anti-Ly6C (HK1.4, Thermo Fisher) and Alexa Fluor 700-anti-CD11c (N418, Thermo Fisher) antibodies were used. Isotype control antibodies were used optimally.
All samples were acquired and analysed using a FACSAria™ III flow cytometer (BD Biosciences) and a BD Accuri C6 flow cytometer (BD Biosciences).

Mice were administered with 2 × 10⁹ BLP-FITC orally or nasally and euthanized at 6 or 24 h post-administration. Single cell suspensions from the spleen were prepared as above. To stain dead cells, cells were treated with APC-Cy7-conjugated FVD780 (eBioscience). The cells were then incubated with TruStain FcX™ (anti-mouse CD16/32) Antibody (Biolegend) to block Fc receptor, followed by incubation with PE-conjugated anti-CD11b (M1/70, Biolegend) and APC-conjugated anti-CD11c (N418, Biolegend). The cells were analyzed by FACSverse (BD). At least 2.5 × 10⁵ live singlet cells were used for the analysis.