Fitc conjugated rat igg2b

Splenocytes were collected from glycolipid‐treated mice and diluted with staining solution (PBS (pH = 7.4) containing 0.1% bovine serum albumin). For further analysis of cell subpopulations, an array of antibodies was used for surface staining. All fluorescence‐conjugated antibodies were obtained from BioLegend as follows: FITC‐conjugated CD45 (30‐F11), PE‐conjugated antimouse CD54 (YN1/1.7.4), APC‐conjugated CD3 (145‐2C11), APC/Fire750‐conjugated CD8 (53–6.7), FITC‐conjugated CD4 (GK1.5), Brilliant Violet (BV) 421‐conjugated F4/80 (BM8123137), BV605‐conjugated CD11b (M1/70), APC/Fire750‐conjugated CD11c, PE‐conjugated NK1.1 (PK136), BV510‐conjugated Ly‐6C (HK1.4), and BV785‐conjugated Ly‐6G (1A8). After blocking with TruStain fcX (antimouse CD16/32) at 4 °C for 15 min, 2 × 10⁶ cells were stained with antibodies at 4 °C for 30 min, using 0.2 µg antibody per sample in 50 µL staining solution.
Analogously, isotypes were also applied in FACS analysis, including APC‐conjugated Armenian Hamster IgG, FITC‐conjugated rat IgG2b, APC/Fire750‐conjugated rat IgG2a, PE‐conjugated rat IgG1, PE‐conjugated rat Ig2b, PE‐conjugated mouse IgG2a, APC/Fire750‐conjugated Armenian Hamster IgG, FITC‐conjugated rat IgG2b, APC‐conjugated rat IgG2b, PE‐conjugated rat IgG2a, and PE/cy7‐conjugated rat IgG2b.k (Biolegend).

Anti-mouse CD16/32 antibody was used to block Fc receptors on PLF or spleen cells by treating them with cold for 20 minutes. Single-cell suspensions were stained with a combination of the following antibodies: PerCP-anti-CD11b (M1/70), FITC-anti-Ly6G (1A8), PerCP-anti-CD4 (GK1.5), PE-anti-CD25 (PC61), FITC-anti-Foxp3 (FJK-16s), and APC-anti-CTLA-4 (UC10-4B9) (all from BioLegend). Neutrophils were recognized as CD11b⁺Ly6G⁺, and Tregs as CD4⁺CD25⁺Foxp3⁺. The cells were fixed and permeabilized following the kit’s instructions (BioLegend) before being stained for Foxp3. PerCP-conjugated rat IgG2b, PE-conjugated rat IgG2a, FITC-conjugated rat IgG2b, and APC-conjugated rat IgG1 were all used as isotype controls (all from BioLegend). Fluorescence-activated cell sorting (FACS) analysis was performed using an Accuir C6 Plus cell analyzer (BD Bioscience). Flow cytometry data were analyzed with FlowJo (Tree Star, Inc., Ashland, OR, USA). For all samples, ≥1×10⁵ cells were collected to generate scatter plots.

Cells present in the PLF or obtained from omentum were pretreated on ice for 20 min with anti-mouse CD16/32 abs to block FC receptors. For cell surface staining, cells were stained on ice for 30 min with the following fluorescently conjugated antibodies: peridinin-Chlorophyll-Protein Complex (PerCP)-anti-CD45 (30-F11), phycoerythrin (PE)-anti-CD11b (M1/70), isothiocyanate (FITC)–anti-Ly6G (1A8), PE-anti-CD19 (6D5), and FITC–anti-CD11b(M1/70) (all from BioLegend). Neutrophils were identified by CD11b⁺Ly6G⁺, and B1 cells were identified by CD19⁺CD11b⁺. For intracellular staining, a cytofix/Cytoperm kit (BD) was used to fixed and permeabilized cells. Allophycocyanin (APC)–anti-TNF-α (MP6-XT22) and APC–anti-IL-6 (MP5-20F3) Ab were used for intracellular staining. Isotype controls used were PerCP-conjugated rat IgG2b, PE-conjugated rat IgG2a, FITC-conjugated rat IgG2b, and APC-conjugated rat IgG1 (all from BioLegend). FACS analysis was performed using a BD Accuir C6 Plus cell analyzer (BD). All data were analyzed using FlowJo (Tree Star). For all samples, at least 5×10⁴ cells were collected to generate scatter plots.