Anti mouse cd11c antibody

Lungs were isolated and pooled from three newborn mice. Lung tissue was cut into pieces and incubated (37 °C, 30 min) with shaking (150 r.p.m.) in digestion buffer (RPMI 1640 with 10% FBS, 15 mM HEPES, 1% penicillin/streptomycin (wt/vol) and 300 U/ml collagenase VIII) and then pressed through a 100-µm nylon strainer to obtain a single-cell suspension. 1 × 10⁷ cells were washed and then incubated (4 °C, 10 min) with anti-mouse CD16/CD32 and then re-incubated (4 °C, 30 min) with anti-mouse CD45 antibody (30-F11), anti-mouse CD4 antibody (GK1.5), anti-mouse CD8 antibody (53-5.8), anti-mouse CD11b antibody (M1/70), anti-mouse CD11c antibody (N418), anti-mouse CD19 antibody (6D5), anti-mouse Ly6G antibody (1A8), anti-mouse F4/80 antibody (BM8) (all diluted 1:100, Biolegend). Cells were sorted using a LSRII (BD Biosciences) and the data analyzed with FlowJo (Treestar).

As previously described [19 (link)], each group's DCs and T cells that were going to be examined in the coculture system were first collected, then washed twice with PBS after being digested with 0.25 percent trypsin. After this, the cells were tested. Following centrifugation, the supernatant was discarded, and the cells were resuspended in Eppendorf (EP) tubes at a concentration of 2 105 cells per 100 μL. Then, l μL of primary antibodies was added to the EP tube, including antimouse CD133 antibody (BioLegend, USA), antimouse CD11c antibody (BioLegend, USA), antimouse CD80 antibody (BioLegend, USA), antimouse CD86 antibody (BioLegend, USA), antimouse MHC-II antibody (BioLegend, USA), antimouse CD4 antibody (BioLegend, USA), antimouse CD8 antibody (BioLegend, USA), and antimouse FOXp antibody (BioLegend, USA). Following a thirty minute incubation period in the dark at a temperature of four degrees Celsius, the cells were resuspended in 0.2 milliliters of PBS before being washed twice with PBS. The phenotypic changes in DCs and T cells were identified with the help of flow cytometry (a BD FACSCalibur), and the analysis of the experiment's results was performed with the FlowJo software tool (FlowJo™ v10.7, http://www.flowjo.com).

Virus infected or uninfected (control) cell lines were blocked with Human TruStain FcX (Biolegend, San Diego, CA). For PBMC staining and CD141 detection, anti-human CD141 (Biolegend), anti-human CD11c (Biolegend), and anti-human lineage cocktail 1 (Lin1) (Biolegend) were used. For SCT detection DCs were stained with anti-human HLA-A2 (BD Pharmigen) antibody. Mouse DCs were identified using anti-mouse CD11c antibody (Biolegend). All FACS data was acquired on FACSCalibur (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (FlowJo, Ashland, OR).