Fitc conjugated anti f4 80

To examine the surface expression of PDPN in mouse tumor cell lines, tumor cells were incubated with PMab‐1‐mG_2a or isotype control (1 μg/mL) for 30 min. Cells were washed with PBS and incubated for 30 min with FITC‐conjugated goat F(ab’)₂ fragment anti‐rat IgG (H + L) Ab (Beckman Coulter). AB1‐HA tumor tissue was cut into small pieces with scissors and digested by digestion buffer consisting of 1 mg/mL BSA (Sigma‐Aldrich), 1 mg/mL collagenase IV (Thermo Fisher Scientific Inc.), and 100 μg/mL DNase I (Roche) in DMEM for 40 min of incubation at 37°C, followed by passaging through a 100 μm cell strainer. The cells were incubated with FcR blocking reagent (BD Biosciences). To examine the surface expression of CTLA‐4 and PD‐1 in mouse tumor‐infiltrating immune cells, tumor tissue‐derived cells were stained with FITC‐conjugated anti‐NKp46 (BioLegend), FITC‐conjugated anti‐CD8a (BD Biosciences), FITC‐conjugated anti‐CD4 (BD Biosciences), FITC‐conjugated anti‐F4/80 (BioLegend), PE‐Cy7‐conjugated anti‐CTLA‐4 (BioLegend), and PE‐Cy7‐conjugated anti‐PD‐1 (BioLegend). The stained cells were analyzed by flow cytometry using a BD LSRFortessa (BD Biosciences) for acquisition and the FlowJo software program (Treestar Inc.) for the analysis.

Flow cytometry was performed using FACS Calibur (BD Biosciences). CT26 cells were cultured with or without 50 ng/mL interferon (IFN)-γ (Tonbo Biosciences) for 48 hours and stained with anti-CCR7 (clone 4B12), anti-PD-L1 (clone 10F.9G2), and isotype-matched control antibodies (BioLegend). iFib, iMSC, and iMSC/CCL19 were stained with the following antibodies as described previously (3): anti-PDGFRα (clone APA5), anti-PDGFRβ (clone APB5), anti-CD34 (clone HM34), anti-Sca-1 (clone D7), and anti-CD29 (HMβ1–1) (all antibodies from BioLegend). Anti-CD44 (clone IM7), anti-CD45 (clone 30-F11), and anti-CD117 (clone ACK2) antibodies were purchased from Tonbo Biosciences. Cell suspensions from tumor or spleen were stained with the following antibodies: PE-conjugated anti-CD3, PerCP-Cy5.5-conjugated anti-CD4, FITC-conjugated anti-CD8α, PerCP-Cy5.5-conjugated anti-CD11c, APC-conjugated anti-CCR7, FITC-conjugated anti-PD-1, and FITC-conjugated anti-F4/80 (antibodies purchased from BioLegend).
ELISA iFib, iFib/CCL19, iMSC, and iMSC/CCL19 cells were cultured for 48 hours. Then, the supernatants were collected, and the levels of CCL19 in the supernatants were measured using the mouse MIP3 beta ELISA Kit (#ab100729, Abcam) and Multiskan FC Basic plate-reader at 450 nm (Thermo Fisher).