Rat igg2a kappa isotype control

Analytical-grade chemicals and reagents were purchased from Sigma (Sigma-Aldrich, Gillingham, UK) if not stated otherwise. Restriction enzymes for cloning were from Fermentas (ThermoFisher, Waltham, MA, USA). Antibodies from the following sources were used for Western blotting: mouse monoclonal anti-human CCL19 (R&D Systems, Minneapolis, MN, USA, #AB361), goat polyclonal anti-human CCL21 (R&D Systems, Minneapolis, MN, USA, #AF457), polyclonal rabbit anti-goat coupled to HRP (Dako Agilent, Santa Clara, CA, USA, #P0160), polyclonal goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA, #115-035-003). For flow cytometry, the following antibodies were used: huCCR7-PacificBlue (Biolegend, San Diego, CA, USA, #353210), huCCR7-APC (R&D Systems, #FAB197A), huCD3-PacificBlue (Biolegend, San Diego, CA, USA, #300417), huCD4-FITC (Bio-Rad, Hercules, CA, USA, #MCA1267F), huCD45RA-FITC (Bio-Rad, Hercules, CA, USA, #MCA88F), huCD45RO-FITC (Bio-Rad, Hercules, CA, USA, #MCA461F), muCD3ε-PE (Biolegend, San Diego, CA, USA, #100307), muCCR7-APC (ThermoFisher, Waltham, MA, USA, #17-1971-82) and rat IgG2a kappa isotype control (ThermoFisher, Waltham, MA, USA, #17-4321-81). Dy^649P1 (#649P1-03) was purchased from Dyomics GmbH (Jena, Germany) and conjugated to CoA as published previously [42 (link)].

T-cell populations were analyzed by flow cytometry using procedures previously described^{46 (link)}. Briefly, MLNs were harvested, homogenized, and resuspended in PBS containing 2% fetal bovine serum. For all samples, Fc receptor blocking was performed with Purified Rat Anti-Mouse CD16/CD32 (BD Biosciences, San Jose, CA, USA). Cells were stained using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) to assess viability. anti-mouse CD45 (BioLegend, San Diego, CA, USA), anti-mouse TCRβ (BioLegend), and anti-mouse CD4 (BioLegend) antibodies were used for surface staining. A FOXP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) was used to fix and permeabilize cells. Anti-mouse/rat Foxp3 (Thermo Fisher Scientific), anti-mouse/human T-bet (Thermo Fisher Scientific), and anti-Mouse RORγt (Thermo Fisher Scientific) antibodies were used for intranuclear staining. Mouse IgG1 Kappa Isotype Control (Thermo Fisher Scientific), Rat IgG1 Isotype Control (Thermo Fisher Scientific), and Rat IgG2a Kappa Isotype Control (Thermo Fisher Scientific) were used as isotype controls. Samples were analyzed with CytoFLEX (Beckman Coulter, Brea, CA, USA) and FlowJo version 10.8 (FLOWJO, Ashland, OR, USA).

Nb119 and NbBCII10 monoclonal antibody were labelled using alexa fluor-647 lightning-link APC conjugation kit (Innova biosciences, San Diego, CA, USA). Non-activated macrophages were harvested from the peritoneal cavity of mice. About 5 × 10⁵ cells were washed three times with PBS-2% FCS and resuspended in a total volume of 100 µL. Anti-F4/80 (Cl:A3-1)/Alexa fluor 488 and Anti-Mouse CD11b (M1/70)/PE antibodies were purchased from AbD Serotec and Ebioscience. Anti-Vsig4 (NLA14) monoclonal antibody and rat IgG2a kappa isotype control were purchased from Ebioscience. Five µg of antibody or nanobody were used per 1 × 10⁶ cells for 20 min at 4 °C. Excess fluorescein labelled antibody was removed by washing with PBS-2% BSA. Stained cells were further analyzed by flow cytometry and histograms were prepared using FlowJo software (Becton Dickinson, San Jose, CA, USA).