Cd86 bv421

Single-cell suspensions of tumors were prepared, and flow cytometry was carried out as mentioned earlier [45 (link), 46 (link)]. Cells were stained with the following antibodies obtained from BioLegend: CD16/32 (clone 93, catalog No. 103132), Zombie UV™ Fixable Viability Kit (catalog No. 100752), CD45-PerCP/Cy5.5 (clone 30-F11, catalog No. 103132), CD45-APC/Cy7 (clone 30-F11, catalog No. 103116), CD11b-FITC (clone M1/70, catalog No. 101206), SIRPα-PE (clone BM8, catalog No. 123122), Gr-1-PerCP/Cy5.5 (clone RB6-8C5, catalog No. 108428), F4/80-AF647 (clone N418, catalog No. 117318),CD11c-PE/Cy7 (clone N418, catalog No. 117318), I-A/I-E-AF700 (clone M5/114.15.2, catalog No. 107622), CD86-BV421 (clone GL-1, catalog No. 105032), CD206-BV605 (clone C068C2, catalog No. 141721), CD8a-AF700 (clone 53-6.7, catalog No. 100730), CD8-BV510 (clone 53-6.7, catalog No. 100752), TNF-α-BV421 (clone MP6-XT22, catalog No. 506328), IFN-γ-PE (clone XMG1.2, catalog No. 505808), CD39-PE/Cy7 (clone Duha59, catalog No. 143806), Tim-3-APC/Fire™ 750 (clone RMT3-23, catalog No. 119738), PD-1-BV605 (clone 29 F.1A12, catalog No. 135220). The next antibodies were obtained through eBioscience: Mouse Regulatory T Cell Staining Kit #2 (clone FJK-16s, catalog No. 88-8118-40). FlowJo program (Tree Star Inc.) was utilized to examine the findings that were acquired through a BD LSRFortessa Flow Cytometer.

Fresh spleen, lung, and tumor tissues were removed from the mice immediately after sacrifice and prepared as single-cell suspensions at 5 × 10⁶/mL. Antibody labeling was performed using Fc-receptor blockers [Purified Rat Anti-Mouse CD16/CD32 antibody (BD, catalog no. 553141, USA), True-Stain Monocyte Blocker (BioLegend, catalog no. 426102, USA)], and Zombie NIR Fixable Viability Kit (BioLegend, catalog no. 423105, USA). The RedFluor™ 710 Anti-Mouse CD45 (30-F-11) (TONBO Biosciences, catalog no. 80–0451, USA), PerCP-Cyanine5.5 anti-Mouse F4/80 antigen (BM8.1) (TONBO Biosciences, catalog no. 65–4801, USA), CD11b monoclonal antibody (M1/70) eFluor 450 (eBioscience, catalog no. 48–0112-82, USA), CD206 (MMR) monoclonal antibody (MR6F3) PE-Cyanine7 (eBioscience, catalog no. 25–2061-82, USA) were applied for staining according to the manufacturer's instructions. In vitro, CD86 BV421 (Biolegend, 105031, GL-1) and anti-PDL1 PE (BD, 558091, MIH5) antibodies were added to the staining scheme. The stained single-cell suspensions were assessed by a BD LSR Fortessa flow cytometer (BD Biosciences, USA) with FlowJo (version 10) software.

Peanut-sensitized mice were intragastrically administered twice daily with PBS, sodium butyrate or ButM at an equivalent dose of 0.224 mg g⁻¹ (butyrate per mouse) for 2 weeks. After the treatment, spleen, ileum and colon-draining LNs from mice were collected, and digested in DMEM supplemented with 5% FBS, 2.0 mg ml⁻¹ collagenase D (Sigma Aldrich) and 1.2 ml CaCl_2. Single-cell suspensions were prepared by mechanically disrupting the tissues through a cell strainer (70 μm, Thermo Fisher). Splenocytes (4 × 10⁶) or cells from LNs (1 × 10⁶) were plated in a 96-well plate. Cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Thermo Fisher), followed by surface staining with antibodies in PBS with 2% FBS, and intracellular staining according to the manufacturer’s protocols for eBioscience Foxp3/Transcription Factor Staining Buffer set (Invitrogen). The following anti-mouse antibodies were used: CD3 APC/Cy7 (clone 145-2C11, BD Biosciences), CD4 BV605 (clone RM4-5, Biolegend), CD25 PE/Cy7 (clone PC61, Biolegend), Foxp3 AF488 (clone MF23, BD Biosciences), CD11b BV711 (clone M1/70, BD Biosciences), CD11c PE/Cy7 (clone HL3, BD Biosciences), F4/80 APC (clone RM8, Biolegend), I-A/I-E (MHCII) APC/Cy7 (clone M5/114.15.2, Biolegend) and CD86 BV421 (clone GL-1, Biolegend). Stained cells were analysed using an LSR Fortessa flow cytometer (BD Biosciences).