Fcr blocker

Flow cytometry was performed as previously published [20 (link)]. Briefly, after harvesting, glioblastoma cells were spun down in Eppendorf tubes and re-suspended in 80 μL buffer and 20 μL FcR blocker (Miltenyi Biotec, Bisley, UK), after which 10 μL anti-CD133/1 (AC133)-PE antibody (Miltenyi Biotec, Bisley, UK) or a mouse monoclonal IgG-PE isotype control was added according to the manufacturer’s instructions. Next, the cells were analysed using a Beckman Coulter flow cytometer. The resulting data were analysed using the Weasel software (http://www.frankbattye.com.au/Weasel/).

Staining of immune cells extracted by ED was performed by blocking the Fc receptor using FcR Blocker (Miltenyi Biotec, Bergisch Gladbach, Germany). 7AAD viability dye (eBioscience) was then added, followed by staining with mouse anti-human CD11b-APC-Cy7 (BD Biosciences), mouse anti-human CD33-FITC (BioLegend, San Diego, USA), mouse anti-human HLA-DR-PE (BD Biosciences), CD14-PE-Cy7 (eBioscience) and mouse anti-human CD15-APC (BioLegend). After incubation at 4 °C for 25 min, samples were washed twice with PBS and the pellets were resuspended in 300 µl flow cytometry staining buffer (eBioscience) and analyzed using BD FACSCanto II flow cytometer. Some tumor-infiltrating immune cells were also stained for ARG1 expression, as described above, with the addition of Fixable Viability Dye eFluor^® 780 (FVD780; eBioscience) to gate live cells. Flow cytometric data were analyzed using BD FACSuite software (BD Biosciences).

Cell surface markers were analyzed using specific florescence conjugated and non-conjugated antibodies. Non-specific florescence was attuned using appropriate Isotype controls. For monocyte and macrophage specific cell surface antigen expression, mouse anti-human- CD68 FITC, CD14 PE, CD11b APC, HLA-ABC FITC, HLA-DR FITC, CD80 FITC, CD86 PE, CD50 FITC, CD54 PE, CD163 PE, CD205 PE and CD206 APC antibody conjugates from BD Pharmingen; CX3CR1 FITC from R&D Systems were used. To prevent non-specific binding of antibodies to macrophage/monocyte lineage cells, cells were pre-incubated with buffer containing 2 mM EDTA, 0.5% FBS. DPBS and 10 μl of FcR blocker (Miltenyi Biotec) for 15 min at 4 °C. Cells were then washed with DPBS and stained in antibody containing FACS buffer (DPBS containing 1% FBS and 0.01% sodium azide) on ice for 1 hour. For indirect staining, cells were further washed and treated with appropriate secondary antibody for 30 minutes on ice. Stained cells were fixed with 1% Para Formaldehyde and stored at 4 °C till further analysis. Cells were acquired on a BD FACS CALIBUR and the data was analyzed using the Cell Quest Pro software. % positivity was calculated for each surface antigen after gating with respect to relevant isotype control antibody.

Cells were adjusted to a concentration of 1 × 10⁶ cells/mL in 100 μl cold PBS, washed with FACS buffer (2% PBS and 0.5 mM EDTA in PBS), and blocked with FcR blocker (Miltenyi) for 20 min at 4 °C, followed by incubation with anti-FAP primary antibody (1:100; Additional file 1: Table S1) for 60 min in dark at 4 C. After 3 washes, cells were incubated with APC-anti-Rabbit IgG (R and D Systems, 1:100) on ice in the dark for 40 min. After 2 washes with FACS buffer, cells were transferred to FACS tubes and subject to cell sorting using a FACS machine (BD). Cells were collected in DMEM, immediately centrifuged at 1500 rpm for 5 min, and the pellets stored at − 80 °C freezer for later RNA extract.

Single-cell suspensions from mouse spleen and joint tissues were prepared. A Zombie NIR Fixable Viability Kit (BioLegend) staining was performed for eliminating dead cells. Gated living cells were analyzed. Surface staining, such as anti-CD4 and isotype IgG control, was performed for 20–30 min. For intracellular staining, harvested cells were stimulated for 4–6 h in culture medium with PMA (Sigma-Aldrich), ionomycin (Sigma-Aldrich), and monensin (Sigma-Aldrich). Fixation/Permeabilization (eBioscience) was used for fixation and permeabilization, followed by Fc-R blocker (Miltenyi) and staining with the appropriate antibodies, including anti-IL-17A, anti-IFNγ, anti-Rorγt and their isotype IgG controls. Absolute cell numbers were calculated on the basis of the percentage of each cell population. All procedures were performed according to the manufacturer’s instructions.

Sixty-eight hours after the 3rd nasal immunization, NALT cells were prepared from mice as described. After being blocked with FcR blocker (Milteny Biotec, Bergisch Gladbach, Germany), the cells were stained with fluorescein isothiocyanate- (FITC-) labeled anti-mouse CD11c antibody and FITC-labeled anti-mouse CD80 antibody, or FITC-labeled anti-mouse CD86 antibody (Milteny Biotec), and subjected to flow cytometry (FCM) analysis using a flow cytometer (GALLIOS, Beckman Coulter, Brea, CA).