Anti f4 80 fitc

Joint macrophages were prepared and filtered with a cell strainer. F4/80 macrophages were used as pan-macrophage markers, iNOS was used as a marker of M1 macrophages and CD206 was used as a marker of M2 macrophages [18 (link),31 (link),32 (link)]. Surface staining was performed using the following monoclonal antibodies: anti-F4/80 FITC (eBioscience), anti-CD206 PE (BioLegend, San Diego, CA, USA). Anti-iNOS allophycocyanin was purchased from BD Biosciences. For intracellular staining of iNOS, Cell Stimulation Cocktail (a cocktail of phorbol 12-myristate 13-acetate, ionomycin, brefeldin A and monensin from eBioscience) was added and cultured for the last 5 hours before flow cytometric analysis as previously described [25 (link)].

Immediately after sacrifice, the liver tissues were excised and washed with PBS to remove blood. Tissues were minced and digested with 1 mg/mL collagenase type IV (Sigma‒Aldrich) in PBS containing 150 U/mL DNase I for 30 min at 37 °C. The digested liver was suspended in 50 mL of DMEM, and the dissociated cells were filtered through a 100-μm nylon strainer and collected by centrifugation at 300 × g for 4 min. Stromal vascular fraction pellets were resuspended in 0.5 ml of RBC lysis buffer (BioLegend) for 5 min on ice, centrifuged for 5 min at 300 × g, and resuspended in PBS. Cells were incubated with the following fluorescently labeled antibodies for 30 min: anti-F4/80-FITC (eBioscience, 11-4801-82, 1:10) or anti-CD11b-PE (eBioscience, 12-0112-82).
Isolation of LPCs was performed as previously described^{21 (link),26 (link)}. Red cells were lysed with buffer (160 mM NH₄Cl, KHCO₃ containing 0.01% EDTA) and resuspended in Williams E medium (Gibco) with 10% FCS. Following centrifugation at 300 × g for 5 min at 4 °C, the supernatant was collected, washed twice, and resuspended in PBS with 2% FCS for FACS staining. LPCs were stained with anti-LGR5 (Abnova, PAB2591, 1:10) at 4 °C for 30 min and then incubated with Alexa 555-conjugated IgG secondary antibody (Invitrogen, A-21428) at 4 °C for 10 min. The stained cells were analyzed by flow cytometry (BD Bioscience).

Isolated bone marrow cells from 3 month old mice and were blocked for non-specific staining in 2% BSA and with anti-CD16 (5 μ g/mL) for 20′ at 4°C. Afterwards, cells were stained in 2% BSA with anti-CD4-Violet421 (1:100, Pharminogen), anti-CD8-PE-Cy7 (1:100, Biolegend), anti-F4/80- Fitc (1:100, eBiosciences), anti-CD3-e450 (eBiosciences), anti-NK1.1- PE-Cy7 (eBiosciences), anti-CD19-Violet421 (1:100, Biolegend) and anti-B220-PerCP (1:100, Biolegend), anti-CD11c-Violet 421 (1:100, Biolegend), anti-MHCII-PerCP (1:100, Biolegend). At last, the cells were washed twice in 2% BSA and analyzed by flow cytometry using Attune (Invitrogen), and FlowJo.

TAME was performed as previously described^{31 (link), 32 (link)} in HLA-A2.1 transgenic mice. Briefly, single-cell suspensions of pooled spleen and lymph nodes were stained with WT1A-PE, WT1B-APC or both tetramers before magnetic enrichment. Mice were randomly divided into groups of five. Enriched cells were then stained with mouse anti-CD3 PerCP-Cy5.5, anti-CD8 APC-Cy7 (#557654, BD Biosciences), anti-CD44 PE-Cy7 (#25044182, eBiosciences), anti-CD62L BV570 (#104433, Biolegend), anti-CD4 FITC (#553055, BD Biosciences), anti-B220 FITC (#553088, BD Biosciences), anti-F4/80 FITC (#11480185, eBiosciences), anti-CD11b FITC (#11011282, eBiosciences), anti-CD11c FITC (#11011482, eBiosciences), anti-I-A^b FITC (#116406, Biolegend) and LIVE/DEAD Fixable Aqua before acquiring using the LSR Fortessa II.

Freshly isolated liver MNCs and spleen cells were initially incubated with anti-mouse CD16/32 (1:100 final dilution; Becton Dickinson) for 15 minutes at room temperature. The washed cells were incubated for 30 minutes at 4 °C with the following fluorescently labeled monoclonal antibodies: anti-ST2L-PE (eBioscience, San Diego, CA), anti-CD11b-APC (eBioscience), anti-CD19-PE-Cy7 (eBioscience), anti-CD4-PerCP (eBioscience), anti-CD3-APC (BD Pharmingen, San Diego, CA), anti-F4/80-FITC (eBioscience), anti-F4/80-PerCP-Cy5.5 (eBioscience), anti-MHCII-FITC (eBioscience). FACS analysis was performed using FACScalibur and/or FACSverse (both from Becton Dickinson).

CD11c⁺, CD11b⁺, and F4/80⁺ were isolated from a suspension of total spleen cells in PBS supplemented with 2 mM EDTA (Sigma-Aldrich, Poznan, Poland) and 0.5% BSA (Sigma-Aldrich) according to the producer's instructions. We used a magnet, columns, and magnetically labeled antibodies from Miltenyi Biotec. In the case of F4/80⁺ cells we performed indirect staining with anti-F4/80 FITC (eBioscience, Vienna, Austria) and anti-FITC magnetic beads, whereas CD11c⁺ and CD11b⁺ cells were labeled directly. CD4⁺ T cells were isolated with a kit for negative selection (depletion of non-CD4⁺ cells). In the case of splenic APCs, we sorted CD11c⁺ at the beginning, next F4/80⁺ cells, and CD11b⁺ cells at the end. In all cases the purity of sorted cells was 85% or higher, as determined by flow cytometry.