Control rat igg

The mice were anesthetized with either isoflurane or 2,2,2 tribromoethanol and were infected intranasally with 10⁶ plaque forming units (PFU) of M2SR virus in 30 μL of sterile PBS. The mock-infected control mice received 30 μL of PBS alone. For comparison, in some experiments the mice were vaccinated with 1 μg of purified inactivated whole influenza A/PR/8/34 (H1N1) antigen (Charles River Avian Vaccine Services, North Franklin, CT, USA) intramuscularly. At specified intervals after immunization, the sera were collected for an antibody titer or the mice were challenged intranasally with 40 mouse-lethal doses of 50 (MLD₅₀) PR8 or 40 MLD₅₀ Aichi. In some experiments, the T cells were depleted from groups of either wildtype C7BL/6 mice or B-cell-deficient muMT mice, using 0.5mg per mouse of each antibody, GK1.5 anti-CD4, 2.43 anti-CD8 and 30H12, anti-Thy1, or 1.5 mg control rat IgG (BioXcell, Lebanon, NH, USA) 3 times per week for 2 weeks prior to the challenge. This protocol depleted >99% of the T cells. Their survival was monitored for 14–21 days after the challenge.

In vivo depletion of Treg cells with anti-CD25 antibodies was performed as previously described [9 (link)]. We verified that this schedule was very effective in the depletion of Treg cells without causing significant alterations in other T cell subsets. Briefly, C57BL/6 Foxp3^GFP mice were given i.p. injections of 500 μg of anti-CD25 (clone PC61) or control rat IgG (BioXcell, USA) diluted in sterile PBS. Antibodies were administered on days -3 and +3 relative to infection with P. brasiliensis yeasts.

RAW264.7 macrophages’ ability to adhere to human-derived lymphatic endothelial cells (HDLECs) or integrate into lymphatic vessel structures following stimulation with rmCHI3L1 was evaluated using a 2D adhesion and 3D integration assay, respectively. Therefore, RAW264.7 macrophages, cultured as previously described [14 (link)], were pretreated ON with 5 μg/ml rmCHI3L1 (Bio-techne), in addition of 0.6 μg/ml anti-PDPN (clone pMAB, Thermo Fisher Scientific) or 0.6 μg/ml rat IgG control (BioXCell, Lebanon, USA), 2 µg/ml IL13Rα2 (polyclonal, Bio-techne) or 2 µg/ml goat IgG control (Bio-techne). The treated RAW264.7 macrophages were fluorescently stained the following day with CellTracker (Thermo Fisher Scientific) and seeded on a HDLEC monolayer for the 2D adhesion assay or together with HDLECs on top of a 4 mg/ml Matrigel pad for the 3D integration assay as previously described [28 (link)]. Images were taken after 4 h for the 2D adhesion assay, and after 7 h for the 3D integration assay.

Neonatal C57BL/6J (wild type), Klrk1-/- (B6.Cg-Klrk1^tm1Dhr/J, Nkg2d-/-) and Tcrd-/- (B6.129P2-Tcrd^tm1Mom/J) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Wild type and knock out mice were inoculated with 3μg/10μl of LPS from E. coli O26:B6 (Sigma-Aldrich, St. Louis, MO), or endotoxin-free PBS (Sigma-Aldrich), intranasally on day of life (DOL) 3, 5, 7, and 10 as described (28 (link)). Selected mice were injected intraperitoneally with 0.5 mg/kg of anti-IL17a antibody (clone TC11-18H10, BioLegend, San Diego, CA) or an IgG control (Rat IgG control, BioXCell, Lebanon, NH) on DOL 3, 4, 5, 6, 7, 10 and 12. Some mice were injected intraperitoneally with anti-NKG2D antibody (Clone HMG2D) or IgG control (10mg/kg) (BioXCell) 1 hr prior to each LPS treatment.